WO2024068997A2 - Antisense oligonucleotides for the treatment of canavan disease - Google Patents

Antisense oligonucleotides for the treatment of canavan disease Download PDF

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WO2024068997A2
WO2024068997A2 PCT/EP2023/077175 EP2023077175W WO2024068997A2 WO 2024068997 A2 WO2024068997 A2 WO 2024068997A2 EP 2023077175 W EP2023077175 W EP 2023077175W WO 2024068997 A2 WO2024068997 A2 WO 2024068997A2
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antisense oligonucleotide
seq
nucleotides
nat8l
target
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Tue FRYLAND
Marianne LERBECH JENSEN
Søren VESTERGAARD RASMUSSEN
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Contera Pharma A/S
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01017—Aspartate N-acetyltransferase (2.3.1.17)

Definitions

  • the present invention relates to antisense oligonucleotides (oligomers) complementary to NAT8L (N-acetyltransferase 8 like) pre-mRNA and mRNA and which are capable of inhibiting the expression of NAT8L. Inhibition of NAT8L expression is beneficial for the treatment of Canavan disease.
  • ASPA encodes aspartoacylase, an oligodendroglial enzyme required for cleavage of the abundant brain amino acid N-acetyl-l-aspartate (NAA) to acetate and L-aspartate.
  • NAA N-acetyl-l-aspartate
  • Canavan disease is a rare and fatal autosomal recessive degenerative disorder that causes progressive damage to nerve cells and pathology of the white matter in the brain. It is one of the most frequent degenerative brain diseases of infancy.
  • Canavan disease belongs to a group of genetic diseases known as leukodystrophies, referring to defects in the development and growth of white matter. Early symptoms of Canavan disease such a hypotonia, macrocephaly, poor head control usually become apparent between the age of 3 and 6 months. The affected infants show delayed developmental milestones (e.g., sitting, standing) and usually never develop the ability to walk. The progressive nature of the disease results in loss of previously acquired abilities and additional symptoms such as seizures and mental retardation also become apparent during infancy. The prognosis for Canavan disease is poor and, in most cases, death occurs before the age of ten.
  • Canavan disease is caused by mutations in the in the ASPA gene, which encodes aspartoacyclase, an enzyme required conversion of the N-acetyl-l-aspartate (NAA) into acetate and L-aspartate.
  • NAA N-acetyl-l-aspartate
  • the ‘Oligodendroglial starvation’ hypothesis proposes that the lack of functional aspartoacyclase results in a deficiency of NAA-derived acetate required for the biosynthesis of myelin (white matter material) and as a result degeneration of myelin/white matter in patients.
  • NAA toxicity proposes, that the excessive NAA concentration in the brain of patients lacking aspartoacyclase, causes an impaired osmolar homeostasis and as result the astroglial and myelinic vacuolation, which is hallmark of Canavan disease (Pleasure et al., Neurochem Res. 2020 Mar;45(3):561-565. doi: 10. 1007/sl 1064-018-2693-6. Epub 2018 Dec 8. Pathophysiology and Treatment of Canavan Disease.).
  • N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time). Moreover, mice with only one intact Nat81 allele (Aspa nur7/nur7 / Nat81 /+ ) accumulated less NAA and developed a less severe disease pathology showed phenotypic improvements and normal lifespan. Similar results have been reported by Sohn et al. (J Neurosci. 2017 Jan 11;37(2):413-421. doi: 10.1523/JNEUROSCI.2013-16.2016. Suppressing N-Acetyl-1- Aspartate Synthesis Prevents Loss of Neurons in a Murine Model of Canavan Leukodystrophy).
  • Hull et al., 2020 showed that antisense oligonucleotides reducing Nat81 expression in Aspa nur7/nur7 likewise reduced levels of NAA, reversed ataxia and reduced the cerebellar and thalamic vacuolation in young-adult Aspa nur7/nur7 mice (Ann Neurol. 2020 Mar;87(3):480-485. doi: 10.1002/ana.25674. Epub 2020 Jan 22. Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice).
  • the present invention provides an antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in a NAT8L (N- acetyltransferase 8 like) gene.
  • NAT8L N- acetyltransferase 8 like
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of the target sequences shown in Table B 1 or DI in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 81 and SEQ ID NO: 882 to SEQ ID NO: 906.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 30 to SEQ ID NO: 81.
  • the sequences are shown in Table Bl in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 11 to SEQ ID NO: 29.
  • the sequences are shown in Table B2 in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 883, 892, 894, 895, 896, 897, 898 or 899.
  • target sequences consisting of SEQ ID NO: 883, 892, 894, 895, 896, 897, 898 or 899. The sequences are shown in Table D2 in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 10.
  • the sequences are shown in Table B3 in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 883, 894, 895, 897, 898 and 899.
  • the sequences are shown in Table D3 in the Examples section.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 4.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 5.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 6.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 7.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 8.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 9.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 10.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 883.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 894.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 895. In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 897.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 898.
  • the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 899.
  • the antisense oligonucleotide comprises a stretch of at least 12, or more preferably at least 14 nucleotides which is at least 90% complementary to a target sequence as set forth herein.
  • the target sequence comprises or consists of a nucleic acid sequence as shown in a sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 402 and SEQ ID NO: 907 to SEQ ID NO: 936.
  • the stretch is 95%, in particular 100% complementary to the target sequence.
  • the antisense oligonucleotide has a length of 12 to 30 nucleotides, more preferably, a length of 14 to 22 nucleotides, and most preferably a length of 16 to 20 nucleotides.
  • the antisense oligonucleotide comprises or consists of a nucleic acid sequence as shown in SEQ ID NO: 403 to SEQ ID NO: 881, such as in SEQ ID NO: 403 to SEQ ID NO: 723, or in SEQ ID NO: 724 to SEQ ID NO: 881.
  • the antisense oligonucleotide is an antisense compound as shown in Table Al in Figure 1, wherein
  • Adx represents 2'deoxyadenosine-3' -phosphorothioate
  • Aox represents 2'-O-methyladenosine-3'-phosphorothioate
  • Amx represents 2'-O-Methoxyethyladenosine-3' -phosphorothioate
  • Alx represents 2'-O-beta-D-oxy LNA adenosine-3' -phosphorothioate
  • Cdx represents 2'deoxycytidine-3' -phosphorothioate
  • Cox represents 2'-O-methylcytidine-3 '-phosphorothioate
  • Cmx represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphorothioate
  • Clx represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphorothioate
  • Edx represents 2'deoxy-5-methylcytidine-3' -phosphorothioate
  • Emx represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphorothioate
  • Elx represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphorothioate
  • Gdx represents 2'deoxyguanosine-3' -phosphorothioate
  • Gox represents 2'-O-methylguanosine-3 '-phosphorothioate
  • Gmx represents 2'-O-Methoxyethylguanosine-3' -phosphorothioate
  • Glx represents 2'-O-beta-D-oxy LNA guanosine-3' -phosphorothioate
  • Tdx represents 2'deoxythymidine-3' -phosphorothioate
  • Tmx represents 2'-O-Methoxyethylthymidine-3' -phosphorothioate
  • Tlx represents 2'-O-beta-D-oxy LNA thymidine-3' -phosphorothioate
  • Uox represents 2'-O-methyluridine-3'-phosphorothioate
  • Ado represents 2'deoxyadenosine-3' -phosphate
  • Aoo represents 2'-O-methyladenosine-3 '-phosphate
  • Amo represents 2'-O-Methoxyethyladenosine-3' -phosphate
  • Alo represents 2'-O-beta-D-oxy LNA adenosine-3' -phosphate
  • Cdo represents 2'deoxycytidine-3' -phosphate
  • Coo represents 2'-O-methylcytidine-3 '-phosphate
  • Cmo represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphate
  • Elo represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphate
  • Edo represents 2'deoxy-5-methylcytidine-3' -phosphate
  • Emo represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphate
  • Elo represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphate
  • Gdo represents 2'deoxyguanosine-3' -phosphate
  • Gmo represents 2'-O-Methoxyethylguanosine-3' -phosphate
  • Gio represents 2'-O-beta-D-oxy LNA guanosine-3' -phosphate
  • Tdo represents 2'deoxythymidine-3' -phosphate
  • Tmo represents 2'-O-Methoxyethylthymidine-3' -phosphate
  • Tlo represents 2'-O-beta-D-oxy LNA thymidine-3' -phosphate
  • Uoo represents 2'-O-methyluridine-3'-phosphate
  • Ad represents 2'deoxyadenosine-3'
  • Ao represents 2'-O-methyladenosine-3'
  • Al represents 2'-O-beta-D-oxy LNA adenosine-3'
  • Cd represents 2'deoxycytidine-3'
  • Em represents 2'-O-Methoxyethyl-5-methylcytidine-3'
  • Gd represents 2'deoxyguanosine-3'
  • Gm represents 2'-O-Methoxyethylguanosine-3'
  • G1 represents 2'-O-beta-D-oxy LNA guanosine-3'
  • Td represents 2'deoxythymidine-3'
  • Tm represents 2'-O-Methoxyethylthymidine-3'
  • T1 represents 2'-O-beta-D-oxy LNA thymidine-3'
  • the antisense oligonucleotide is an antisense compound as shown in Table Cl in Figure 2.
  • the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2 17, 2_35, 2_50, 2_51, 2_52, 6_8, 25 113, 29_2, 29_5, 29_6, 29_7, 29_10, 29_23, 29_24, 29_32, 29_34, 29_36, 29_54, 29_70, 29_72, 29_78, 29_79, 29_84, 29_85, 29_86, 29_100, 29_112, 29_121, 29_123, 29_124, 29_125, 29_130, 29_133, 29_138, 29_154, 34_38, 34_39, 34_46, 34_47, 37 15, 38 5, 38 10, 38 11, or 51 66 as shown in Table Al.
  • the ASO compounds with these ASO IDs had the best effect on the down-regulation of the target gene (see Examples 1 and 2).
  • the antisense oligonucleotide is an antisense oligonucleotide compound selected from the compounds shown in Table A3.
  • the compounds are provided in the so called HELM annotation format.
  • the antisense oligonucleotide is an antisense oligonucleotide compound selected from the compounds shown in Table C3.
  • the compounds are provided in the so called HELM annotation format.
  • the antisense oligonucleotide is an preferably antisense oligonucleotide with ASO ID 2_17, 25 111, 25_113, 29_10, 29_124, 29_130, 29_138, 29_24, 29_34, 29_36, 29_5, 29_70, 29_78, 29_79, 29_84, 29_85, 29_86, 34_39, 34_46, 37 15, 38 10, 51 6, 66_117, 66_120, 66_134, 66_135, 66_123, 66_124, 66_130, 66_126, 66_127, 66_149, 66_189, 66_181, 66_182, 66_173, 66_153, 66_160, 66_185,
  • the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2_17, 25 113, 29_10, 29_124, 29_70, 29_78, 29_79, 29_84, 29_85, 29_86, 34_39, 51_6, 66_120, 66_134, 66_135, 66_124, , 66_130, 66_126, 66_127, 66_149, 66_181, 66_182, 66_173, 66_160, 66_185, 66_183, 66_177, 66_188, 66_140, 66_176, 66_174, 66_220, 66_27, 66_36, 66_39, 66_408, 66_430, 66 431, 66_456, 66_47, 66_485, 66_492, 66_496, 66_545, 66_547, 66_567, 51_6,
  • the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2_17, 29_124, 29_79, 29_84, 29_85, 29_86, 66_134, 66_135, 66_124, 66_130, 66_126, 66_149, 66_181, 66_182, 66_173, 66_160, 66_185, 66_183, 66_177, 66_188, 66_140, 66_176, 66_174, 66_220, 66_27, 66_36, 66_39, 66_408, 66_47, 66_485, 66_496, 66_545, 66_547, 66_567, 66_573, 66_584, 66_587, 66_576, 66_588, 66_63, 66_592, 66_593, 66_600, 66_64, 54_3, 54
  • the antisense oligonucleotide of the present invention shall be capable of reducing the amount of NAT8L (N-acetyltransferase 8 like) mRNA in a host cell expressing said NAT8L mRNA.
  • the host cell is a mammalian cell, such as a primate cell. In a preferred embodiment, said host cell is human host cell.
  • the antisense oligonucleotide is a chemically modified antisense oligonucleotide.
  • a chemically modified antisense oligonucleotide typically comprises modifications of the phosphodiester backbone chemistry, nucleobase modifications and sugar modifications.
  • the chemically modified antisense oligonucleotide comprises at least one 2’ modified sugar or bicyclic sugar.
  • the chemically modified antisense oligonucleotide contains at least one modified nucleobase.
  • at least one modified nucleobase is 5 -methylcytosine.
  • the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2'-O-Methoxyethyl-RNA, 2’-O- Methyl-RNA, 2’-Fluoro-RNA.
  • the chemically modified antisense oligonucleotide may comprise at least one modified intemucleoside linkage. In an embodiment, at least five, such as at least 10 intemucleoside linkages are modified intemucleoside linkages. In an embodiment, all intemucleoside linkages are modified intemucleoside linkages.
  • the antisense oligonucleotide may comprise unmodified intemucleoside linkages (i.e. phosphodiester linkages), modified intemucleoside linkages, or a combination thereof.
  • the modified linkage(s) is (are) selected from: a Phosphorothioate intemucleoside linkage, a Phosphorodithioate intemucleoside linkage, a Phophoroamidate intemucleoside linkage, a methyl phosphonate intemucleoside linkage, a phosphotriester intemucleoside linkage, a boranophosphate intemucleoside linkage and a phosphoryl guanidine intemucleoside linkage.
  • intemucleoside linkage can be stereodefmed versions of said linkages.
  • the at least one modified linkage is a phosphorothioate linkage.
  • at least 50% of the intemucloside linkages, such as all intemucleoside linkages, are phosphorothioate intemucleoside linkages.
  • the antisense oligonucleotide comprises at least one nucleoside with a modified sugar moieity, typically at least four nucleosides with a modified sugar moiety, (herein also referred to as sugar modified nucleosides).
  • the antisense oligonucleotide comprises at least one, such as one, two, three, four or more LNA (locked nucleic acid) or MOE (2’-O-Methoxyethyl) nucleosides.
  • the LNA nucleoside is a beta-D-oxy LNA nucleoside.
  • the antisense oligonucleotide has a gapmer structure, i.e. is a gapmer.
  • the present invention further relates to a conjugate comprising the antisense oligonucleotide according to the present invention, wherein the said antisense oligonucleotide is covalently attached to a conjugate moiety.
  • the present invention further relates to pharmaceutical composition
  • composition comprising the antisense oligonucleotide according to the present invention or the conjugate according to the present invention.
  • the composition further comprises diluents and carriers.
  • the present invention further relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating Canavan disease.
  • the present invention further relates to a method for treating Canavan disease, comprising administering to a subject suffering from Canavan disease a pharmaceutically effective amount of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating Canavan disease.
  • the present invention further relates to a method for identifying a candidate compound for the treatment of Canavan disease, comprising a) providing an antisense oligonucleotide according to the present invention, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
  • Figure 1 Table Al with information on antisense compounds tested in the studies underlying the present invention.
  • Figure 2 Table Cl with information on antisense compounds tested in the studies underlying the present invention.
  • NAT8L N-acetyltransferase 8 like
  • Target regions within the NAT8L pre-mRNA were identified which - when targeted by antisense oligonucleotides - allow for efficient downregulation of the human NAT8L pre-mRNA (or mRNA) in a host cell expressing said pre-mRNA or mRNA (see Tables 3, 6 and 8). Further, down-regulation of expression was observed in a neuronal cell line.
  • the sequences of the target regions are shown in Tables Bl, B2, B3 and DI.
  • the invention provides antisense oligonucleotides which are capable of downregulating NAT8L.
  • the antisense oligonucleotides comprise a stretch of at least 10 nucleotides which is preferably 90%, more preferably, 95% and most preferably fully complementary (i.e. 100% complementary) to the target region (herein also referred to as target sequence).
  • target sequence the target region
  • the antisense oligonucleotides of the present invention are candidates for the treatment of Canavan disease.
  • compounds with a low neuronal toxicity were identified.
  • after administration of a compound of the present invention to a non-human primate no adverse side effects were observed.
  • the present invention relates to an antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in a NAT8L (N- acetyltransferase 8 like) gene.
  • NAT8L N- acetyltransferase 8 like
  • oligonucleotide as used herein is well known in the art. As used herein, the term refers to a molecule of at least ten covalently linked nucleotides. Typically, the oligonucleotides as referred to herein are chemically synthesized, for example by solid-phase chemical synthesis. The oligonucleotides as referred to herein shall contain various chemical modifications which typically do occur in nature. For example, the antisense oligonucleotide may contain at least one 2’ modified sugar. In a preferred embodiment, the antisense oligonucleotides are gapmers.
  • the oligonucleotides of the present invention are antisense oligonucleotides, and in particular single-stranded oligonucleotides. Accordingly, they shall be capable of binding the NAT8L gene, in particular to the NAT8L pre-mRNA, when expressed in a cell, thereby down-regulating the expression of NAT8L gene in the cell.
  • the cell is a human cell is a cell of the central nervous system (CNS).
  • the cell is a brain cell.
  • the NAT8L (N-acetyltransferase 8 like) gene is well known the art.
  • the NAT8L gene is typically the human NAT8L gene.
  • Information on the gene, such as on the nucleic acid sequence, can be found in the known databases, for example, under NCBI Gene ID: 339983).
  • Alternative names of the gene are FLJ37478, Hcml3, Shaft or N-acetylaspartate synthetase gene)
  • the human NAT8L gene encodes a protein having N-acetylaspartate synthetase activity (EC 2.3.1.17). Accordingly, it catalyzes the synthesis of N-acetylaspartate acid (NAA) from L-aspartate and acetyl-CoA.
  • NAA N-acetylaspartate acid
  • the protein sequence can be assessed in the Uniprot database under the accession number Q8N9F0 (NAT8L_HUMAN).
  • the human NAT8L protein has an amino acid sequence as shown in SEQ ID NO: 3 (which is encoded by a transcript having a sequence as shown in SEQ ID NO: 2).
  • N-acetyltransferase 8 like Alternative names are N- acetylaspartate synthetase, NAA Synthetase, or aspartate N-acetyltransferase).
  • the ASO of the present invention targets the human NAT8L pre-mRNA, i.e. downregulates expression of said pre-mRNA.
  • the sequence of the human NAT8L pre-mRNA can be e.g. assessed in the Ensembl database under accession number in ENST00000423729.3. It is encoded by a region on human Chromosome 4: position 2,059,327-2,069,089 on the forward strand (Assembly GRCh38).
  • the sequence of the human pre-mRNA has a sequence as shown in SEQ ID NO: 1.
  • the pre-mRNA is further processed, i.e., by splicing, thereby generating a protein coding mRNA (herein also referred to as transcript).
  • the antisense oligonucleotide of the present invention may also target the human NAT8L mRNA (if the target region is located within an exon, either coding or in the 3’- or 5’UTR).
  • the antisense oligonucleotide of the present invention may target the human NAT8L mRNA having a sequence as shown in SEQ ID NO: 3.
  • SEQ ID NO: 1 and 3 are RNA sequences. In the sequence listing, they are provided as DNA sequences.
  • target RNA sequences have uracil (U) bases instead of thymidine bases (T).
  • U uracil
  • T thymidine bases
  • the human NAT8L pre-mRNA comprises three exons and two introns.
  • An overview on the location of the introns, the exon, and the 3 ’ and 5 ’ UTR with the pre-mRNA sequence can be found in the following table.
  • the antisense oligonucleotides of the present invention shall be capable of downregulating, i.e. reducing expression of the NAT8L mRNA in a cell that expresses said mRNA.
  • the expression is reduced in a call by antisense oligonucleotides of the present invention by least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a control cell (i.e. an untreated control cell).
  • a control cell i.e. an untreated control cell.
  • How to assess whether the expression is reduced can be assessed by well-known methods, i.e. by measuring the expression level (i.e. the amount of the target mRNA) in ASO treated cells.
  • the down-regulation of the target gene is assessed as described in the Examples section.
  • As control for down regulation untreated cells can be used.
  • Down-regulating the expression of the NAT8L mRNA typically, leads downregulation of the NAT8L protein and thus to reduced levels of N-acetyl-L- Aspartate (NAA) as compared to a control.
  • NAA N-acetyl-L- Aspartate
  • Down-regulation of the NAT8L protein can be assessed by e.g. assessing the N-acetylaspartate synthetase activity in cells treated with the ASO of the present invention by using well known enzymatic assays or by or quantifying the protein expression, such as by Western Blotting, mass spectrometry or ELISA.
  • the N-acetylaspartate synthetase activity is reduced in a cell by antisense oligonucleotides of the present invention by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a control cell (i.e. an untreated control cell).
  • the above assessments can be done in vivo or in vitro. If they are done in vitro, they are typically done in human cells, such as in human cells used in the Examples section. In vivo, it is e.g. envisaged that a down-regulation of the target mRNA or protein, of at least 30%, such as at least 40% is achieved. For example, the down-regulation of the target mRNA or protein may be between 40% to 60%, or between 50% to 60% as compared to a control.
  • target sequences within the NAT8L pre-mRNA were identified which can be efficiently targeted with ASOs. In total, 77 of such target regions/sequences were identified. 52 of these target regions are shown in the following table. In the table, each identified target region was assigned a so called “Target ID” (Target ID 1 to 52). These IDs are used throughout the application.
  • Target ID target region
  • target sequence target sequence
  • target nucleic acid are used interchangeably herein.
  • Table Bl in the Example 4 e.g. the sequence of the target region or the SEQ ID NO.
  • Tables B2 and B3 in Example 4 list preferred target regions.
  • the target regions in Tables B2 and B3 may be present in the target regions shown in Table Bl, but may be shorter.
  • Target ID 53 to 77 Some of the target regions shown in the following Table correspond to the target regions in the above table.
  • the target region with Target ID 53 is a subregion of the target region with Target ID 2, i.e. it is contained in this region.
  • the antisense oligonucleotide of the present invention is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in the above table or in Table B 1 in Example 5.
  • the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 30 to SEQ ID NO: 81.
  • the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table B2.
  • the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 11 to SEQ ID NO: 29.
  • the antisense oligonucleotide is capable of binding (i.e. hybridizing, i.e. complementary) to a target region selected from a target region shown in Table B3.
  • the target sequence has a sequence selected from the sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 10.
  • the antisense oligonucleotide of the present invention is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in the above table or in Table D 1 in the Examples section.
  • the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 882 to SEQ ID NO: 906.
  • the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table D2.
  • the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 883, 892, 894, 895, 896, 897, 898 and 899.
  • the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table D3.
  • the target sequence has a sequence selected from the sequences consisting of SEQ ID NO: 883, 894, 895, 897, 898 and 899.
  • the antisense oligonucleotide typically comprises stretch of at least 10 nucleotides which is at least 90% complementary (such as 95% or 100%) to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 81 and SEQ ID NO: 882 to SEQ ID NO: 906.
  • the target sequence has a sequence as shown in SEQ ID NO: 4.
  • the target sequence has a sequence as shown in SEQ ID NO: 5. In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID NO: 6. In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 7.
  • the target sequence has a sequence as shown in SEQ ID NO: 8. In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 9. In another preferred, embodiment, the target sequence has a sequence as shown in SEQ ID NO: 10. In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID: 894. In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID: 883. In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 895.
  • the target sequence has a sequence as shown in SEQ ID NO: 897.
  • the target sequence has a sequence as shown in SEQ ID NO: 898.
  • the target sequence has a sequence as shown in SEQ ID NO: 899.
  • the target sequence preferably comprises a sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 402.
  • the target sequence comprises a sequence selected from the group consisting of SEQ ID NO: 907 to SEQ ID NO: 936 (see Table D4).
  • the target sequences consists of a sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 402, and SEQ ID NO: 907 to SEQ ID NO: 936.
  • the antisense oligonucleotides of the present invention are preferably singlestranded antisense oligonucleotides.
  • the antisense oligonucleotides of the present invention are not inhibitory RNAs.
  • the antisense oligonucleotides of the present invention are not siRNAs or short-hairpin RNAs.
  • the antisense oligonucleotides of the invention shall comprise a “stretch of nucleotides” which is sufficient complementary to a target sequence as referred to herein.
  • the stretch of nucleotides is at least 90% complementary to a target sequence.
  • the stretch of nucleotides is at least 95% complementary to a target sequence.
  • the stretch of nucleotides is fully complementary (i.e. 100% complementary to the target sequence).
  • complementary is well known in the art.
  • the percentage of complementary is typically calculated by calculating the proportion of nucleotides (in %) within the stretch of oligonucleotides of the ASO of the present invention which are complementary to the target sequence within the NAT8L gene.
  • a nucleotide present in the ASO of the present invention are considered as complementary if it forms a Watson-Crick base pair with the nucleotide present in the target RNA sequence.
  • Watson Crick base pairs are guanine-cytosine; adenine -uracil, and adenine- thymine, i.e. G-C, A-U or A-T.
  • modified nucleotides have also the capacity to form such base pairs. For more information, see e.g. Table A2.
  • the “stretch of nucleotides” as referred to herein needs to have a certain length in order to allow for the binding of the oligonucleotide of the present invention to the target region.
  • the stretch of nucleotides has a length of at least 10 nucleotides, more preferably of at least 12 nucleotides and most preferably of at least 14 nucleotides.
  • the antisense oligonucleotide of the present invention may comprise further nucleotides - i.e. in addition to the stretch of nucleotides as referred to above, such as linker nucleotides. These further nucleotides may be complementary to the target sequence, or not.
  • the antisense oligonucleotide of the present invention preferably, has a length of 12 to 30 nucleotides, more preferably, of 14 to 22 nucleotides, and most preferably of 16 to 20 nucleotides. Accordingly, it is envisaged that the antisense oligonucleotide in not longer than 30 nucleotides. In some embodiments, the antisense oligonucleotide in not longer than 22 nucleotides or 20 nucleotides.
  • the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 403 to SEQ ID NO: 723. In another preferred embodiment, the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 403 to SEQ ID NO: 723.
  • the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 724 to 881. In another embodiment, the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 724 to 881.
  • the antisense oligonucleotide comprises or consists of a nucleic acid sequence selected from SEQ ID NO: 724 to 881.
  • the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 410, 517, 558, 580, 582, 584, 661, 557, 570, 573, 613, 614, 621, 630, 707, 725, 799, 801, 802, 803, 805, 785, 787, 819, 822, 823, 789, 828, 829, 830, 831, and 835.
  • the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 410, 517, 558, 580, 582, 584, 661, 557, 570, 573, 613, 614, 621, 630, 707, 725, 799, 801, 802, 803, 805, 785, 787, 819, 822, 823, 789, 828, 829, 830, 831, and 835.
  • the antisense oligonucleotide comprises, preferably, at least 10, more preferably at least 12, even more preferably at least 14 and most preferably, at least 15 consecutive nucleotides of the sequences selected from SEQ ID NO: 403 to 881. Moreover, it is envisaged that the antisense oligonucleotide comprises, preferably, at least 10, more preferably at least 12, even more preferably at least 14 and most preferably, at least 15 consecutive nucleotides of the compounds shown in Table Al and Cl.
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 410 (see e.g. compound with ASO ID 2 17).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 413 (see e.g. compound with ASO ID 2_50, 2_52, or 2_35).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 517 (see e.g. compound with ASO ID 25 111 or 25 113). In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 557 (see e.g. compound with ASO ID 29 5).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 558 (see e.g. compound with ASO ID 29 10).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 561 (see e.g. compound with ASO ID 29 34).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 570 (see e.g. compound with ASO ID 29_70 ).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 573 (see e.g. compound with ASO ID 29_78, 29_79, 29_84, 29_85 or 29_86).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 580 (see e.g. compound with ASO ID 29_124 or 29 121).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 582 (see e.g. compound with ASO ID 29_130).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 584 (see e.g. compound with ASO ID 29 131).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 613 (see e.g. compound with ASO ID 34 39).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 614 (see e.g. compound with ASO ID 34 46).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 621 (see e.g. compound with ASO ID 37 15).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 630 (see e.g. compound with ASO ID 38 10).
  • the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 707 (see e.g. compound with ASO ID 51 6).
  • the antisense oligonucleotide of the present invention is a chemically modified antisense oligonucleotide. Accordingly, it does not occur in nature.
  • a wide range of chemical modification can be incorporated into an oligonucleotide, such modification are e.g. reviewed in Crooke et al. which herewith is incorporated by reference in its entirety (Stanley T Crooke, Xue-Hai Liang, Brenda F Baker, Rosanne M Crooke. Review J Biol Chem. 2021 Jan- Jun;296. antisense technology: A review), which herewith is incorporated by reference in its entirety.
  • At least one of the nucleotides (herein also referred to a monomer) present in the oligonucleotide comprises a chemical modification. More preferably, at least 30%, such as at least 50% of the nucleotides present in the oligonucleotide comprise a chemical modification. In some embodiments, all of the nucleotides comprise chemical modification. Modifications include modifications of the phosphodiester backbone chemistry (“backbone modifications”), nucleobase modifications and sugar modifications. Preferred chemical modifications are shown in Table A2 and C2, see in particular the column “Nucleotide”. The ASOs of the present invention may comprise such nucleotides.
  • the ASO of the present preferably comprises one or more intemucleoside linkages other than a phosphodiester linkage. More preferably, the antisense oligonucleotide comprises at least five, such as at least ten modified intemucleoside linkages. Most preferably, all intemucleoside linkages are modified linkages.
  • the antisense oligonucleotide comprises at least five, such as at least ten modified intemucleoside linkages, such as Phosphorothioate intemucleoside linkage. Most preferably, all intemucleoside linkages are modified linkages. However, some linkages may be phosphodiester linkages, such as one, up to two, up to three or up to four, up to six, or up to eight phosphodiester linkages.
  • the at least one modified intemucleoside linkage is selected from the group consisting of: at least one Phosphorothioate intemucleoside linkage, at least one Phosphorodithioate intemucleoside linkage, at least one Phophoroamidate intemucleoside linkage, at least one methyl phosphonate intemucleoside linkage, at least one phosphotriester intemucleoside linkage, at least one boranophosphate intemucleoside linkage and at least one phosphoryl guanidine intemucleoside linkage.
  • the at least one modified intemucleoside linkage can be a stereodefined versions of said linkages.
  • the oligonucleotide comprises at least one phosphorodithioate intemucleoside linkage.
  • the oligonucleotide comprises at least one phosphoryl guanidine intemucleoside linkage. Most preferably, all internucleoside linkage are phosphoryl guanidine intemucleoside linkages.
  • the oligonucleotide comprises at least one phosphorothioate intemucleoside linkage. Most preferably, all intemucleoside linkage are phosphorothioate linkages.
  • the backbone may be modified with Morpholino Phosphorodiamidate (PMO) and Peptide Nucleic Acid (PNA).
  • PMO Morpholino Phosphorodiamidate
  • PNA Peptide Nucleic Acid
  • Modification applied to the sugar group could be acyclic modifications such as UNA (unlocked nucleic acid), FNA (Flexible nucleic acid), (S)- and (R)-GNA (glycol nucleic acid), D- and L-aTNA (threofiiranosyl nucleic acids) , SNA (Serinol nucleic acids), as described in further details in Bege & Borbas 2021 (Miklos Bege & Aniko Borbas Review Pharmaceuticals (Basel) . 2022 Jul 22;15(8):909. doi: 10.3390/phl5080909. The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic).
  • the chemically modified antisense oligonucleotide comprises one or more modified nucleosides.
  • the one or more modified nucleosides are sugar modified nucleosides, such as one, two, three, four or more sugar modified nucleosides. Typically, it comprises four sugar modified nucleosides.
  • a sugar modified nucleoside is nucleoside with a modified sugar.
  • the one or more sugar modified nucleosides are 2’ sugar modified nucleosides, such as 2’0 modified sugar nucleosides.
  • the 2’0 modified sugar is, selected from the group consisting of 2’-0-Me, 2’MOE (2’- O-Methoxyethyl)), 2’-Npropyl, 2’-O-allyl, 2’F RNA, 2’-O-ethylamine.
  • the 2’0 modified sugar is 2’MOE (2’-O-Methoxyethyl).
  • the modified nucleosides are 2’MOE nucleosides.
  • the one or more modified nucleotides could be locked nucleic acids such as, beta-D-oxy- LNA, 2',4'-constrained 2'-0-ethyl (cEt), such as R-cET and S-cEt, Beta-D-amino LNA, Beta-D-thio LNA, alpha-L-oxy LNA, ENA and other modifications as described in Wan & Seth 2016 (W Brad Wan, Punit P Seth, Review J Med Chem. 2016 Nov 10;59(21):9645-9667.
  • the Medicinal Chemistry of Therapeutic Oligonucleotides preferably comprises one more Locked Nucleic Acid Nucleosides (LNA nucleosides) which are well known 2’- modified nucleosides.
  • LNA nucleosides Locked Nucleic Acid Nucleosides
  • the one or more modified nucleosides are (S)-6’-methyl-beta-D-oxy-LNA (ScET) LNA nucleosides. More preferably, the one or more modified nucleosides are beta-D-oxy-LNA nucleosides,
  • Nucleobase modification include, but are not limited to, 5-methyl-cytosine, pseudo-uridine, 5- Methyluridine, 8-Oxoguanine, 2-thio-thymine, Diaminopurine, abasic nucleosides and others as also described in Brad&Seth 2016 and Robert et al., 2020 (Thomas C Roberts, Robert Langer, Matthew J A Wood. Review Nat Rev Drug Discov. 2020 Oct;19(10):673-694. Advances in oligonucleotide drug delivery)
  • the chemically modified antisense oligonucleotide contains at least one modified nucleobase.
  • the at least one modified nucleobase is 5 -methylcytosine.
  • the ASO may comprise at least pseudouridine, or at least one 8-oxoguanine as modified nucleobase.
  • the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2-O-Methoxyethyl-RNA, 2’-O- Methyl-RNA, 2’-Fluoro-RNA.
  • the antisense oligonucleotide of the present invention has a gapmer structure, i.e. is a gapmer.
  • Gapmers are well known in the art. The term refers to (single stranded) DNA antisense oligonucleotide structures with RNA-like segments on both sides (flanking regions). Gapmers bind to the target sequence and down-regulate target gene expression through the induction of RNase H cleavage.
  • Suitable gapmer designs are well known in the art and are e.g. reviewed in Crooke et al. which herewith is incorporated by reference in its entirety (Stanley T Crooke, Xue-Hai Liang, Brenda F Baker, Rosanne M Crooke. Review J Biol Chem. 2021 Jan-Jun;296. Antisense technology: A review).
  • the gapmer is a LNA gapmer in which the flanking regions comprise LNA nucleosides, such as D-oxy LNA nucleosides.
  • the gapmer may also comprise 2’O-Methoxyethyl (MOE) nucleosides in the flanking regions.
  • MOE gapmers are frequently referred to as MOE gapmers.
  • the antisense oligonucleotide of the present invention has a gapmer structure and at least one modified internucleoside linkage. In a preferred embodiment, the antisense oligonucleotide of the present invention has a gapmer structure and at least 10 modified intemucleoside linkages. In another preferred embodiment, the antisense oligonucleotide of the present invention has a gapmer structure and all linkages are modified intemucleoside linkages. The modified linkages are described herein above. In an embodiment, the modified linkages are phosphorothioate intemucleoside linkages. In another embodiment, the linkages are phosphorodithioates linkages.
  • the antisense oligonucleotide of the present invention is a compound selected from the compounds shown in Table Al (see column “Compound”), wherein
  • Adx represents 2'deoxyadenosine-3' -phosphorothioate
  • Aox represents 2'-O-methyladenosine-3'-phosphorothioate
  • Alx represents 2'-O-beta-D-oxy LNA adenosine-3' -phosphorothioate
  • Cox represents 2'-O-methylcytidine-3 '-phosphorothioate
  • Gox represents 2'-O-methylguanosine-3 '-phosphorothioate
  • Gmx represents 2'-O-Methoxyethylguanosine-3' -phosphorothioate
  • Glx represents 2'-O-beta-D-oxy LNA guanosine-3' -phosphorothioate
  • Tdx represents 2'deoxythymidine-3' -phosphorothioate
  • Tmx represents 2'-O-Methoxyethylthymidine-3' -phosphorothioate
  • Tlx represents 2'-O-beta-D-oxy LNA thymidine-3' -phosphorothioate
  • Ad represents 2'deoxyadenosine-3'
  • Al represents 2'-O-beta-D-oxy LNA adenosine-3'
  • Em represents 2'-O-Methoxyethyl-5-methylcytidine-3'
  • G1 represents 2'-O-beta-D-oxy LNA guanosine-3'
  • Tm represents 2'-O-Methoxyethylthymidine-3'
  • T1 represents 2'-O-beta-D-oxy LNA thymidine-3'
  • Table Al a three letter code or two letter code was used in order to describe the modified nucleotides that are present in the oligonucleotide compounds. Additionally, each of the compounds in Table Al contains a two letter code in the 3’ end, which does not contain the phosphorothioate group. The base and sugar groups of the two letter code is otherwise identical to the three letter. Table A2 provides a translation of the two or three letter codes to their chemical names.
  • HELM “Hierarchical Editing Language for Macromolecules”.
  • the HELM format is described in Zhang T, Li H, Xi H, Stanton RV, Rotstein SH.
  • HELM a hierarchical notation language for complex biomolecule structure representation. J Chem Inf Model. 2012 Oct 22;52(10):2796-806. doi: 10.1021/ci3001925. Epub 2012 Sep 26. PMID: 22947017. The document is herewith incorporated by reference in its entirety.
  • Table A3 shows selected compounds of the present invention in HELM Annotation Format.
  • the antisense oligonucleotide is a compound as shown in Table A3.
  • Table A3 Exemplary compounds of the present invention - HELM Annotation Format
  • [LR](G) is a beta-D-oxy-LNA guanine nucleoside
  • [LR](T) is a beta-D-oxy-LNA thymine nucleoside
  • [LR](A) is a beta-D-oxy-LNA adenine nucleoside
  • [LR]([5meC] is a beta-D-oxy-LNA 5-methyl cytosine nucleoside
  • [dR](G) is a DNA guanine nucleoside
  • [dR](T) is a DNA thymine nucleoside
  • [dR](A) is a DNA adenine nucleoside
  • [dR](C) is a DNA cytosine nucleoside
  • [mR](G) is a 2'-O-methyl RNA guanine nucleoside
  • [mR](U) is a 2'-O-methyl RNA uracil nucleoside
  • [mR](A) is a 2'-O-methyl RNA adenine nucleoside
  • [mR](C) is a 2'-O-methyl RNA cytosine nucleoside
  • [sP] is a phosphorothioate internucleoside linkage.
  • the compound selected from Table Al is a compound which resulted in an efficient downregulation of the target gene in the studies described in Example 1 and/or Example 2.
  • Example 3 The results for the experiments in Example 1 are shown in Table 3.
  • the compound is selected from the compounds resulting in an expression level of NAT8L of 40% or less than 40% , such as of 30% or less than 30% in relation to PBS treated control cells . Information on the expression level can be found in the column “NAT8L PBS norm (A549 High cone)” in Table 3.
  • the results for the experiments in Example 2 are shown in Table 6.
  • the compound is selected from the compounds resulting in an expression level of NAT8L of 50% or less than 50% , such as of 40% or less than 40% in relation to PBS treated control cells . Information on the expression level can be found in the column “NAT8L PBS norm (HEK293 Low cone)” in Table 6.
  • the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2 17, 2_35, 2_50, 2_51, 2_52, 6_8, 25 113, 29_2, 29_5, 29_6, 29_7, 29_10, 29_23, 29_24, 29_32, 29_34, 29_36, 29_54, 29_70, 29_72, 29_78, 29_79, 29_84, 29_85, 29_86, 29_100, 29_112, 29_121, 29_123, 29_124, 29_125, 29_130, 29_133, 29_138, 29_154, 34_38, 34_39, 34_46, 34_47, 37 15, 38_5, 38 10, 38 11, 51 66 as shown in Table Al in the Examples section.
  • the antisense oligonucleotide is a compound selected from the compounds shown in Table A3.
  • the antisense oligonucleotide is the compound with ASO ID 2 17.
  • the antisense oligonucleotide is the compound with ASO ID 2 50.
  • the antisense oligonucleotide is the compound with ASO ID 2 52.
  • the antisense oligonucleotide is the compound with ASO ID 2 35.
  • the antisense oligonucleotide is the compound with ASO ID 25 111.
  • the antisense oligonucleotide is the compound with ASO ID 25 113.
  • the antisense oligonucleotide is the compound with ASO ID 29 5.
  • the antisense oligonucleotide is the compound with ASO ID 29 10.
  • the antisense oligonucleotide is the compound with ASO ID 29 34. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 70. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 78. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 79. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 84. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 85. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 86.
  • the antisense oligonucleotide is the compound with ASO ID 29 124. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 121. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 130. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 138 In another embodiment, the antisense oligonucleotide is the compound with ASO ID 34 39. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 34 46. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 37 15. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 38 10.
  • the antisense oligonucleotide is the compound with ASO ID 51 6.
  • the antisense oligonucleotide of the present invention is a compound selected from the compounds shown in Table Cl in Figure 1 (see column “Compound”).
  • Table Cl a three letter code or two letter code was used in order to describe the modified nucleotides that are present in the oligonucleotide compounds.
  • each of the compounds in Table C 1 contains a two letter code in the 3 ’ end, which does not contain the phosphorothioate group or phosphate. The base and sugar groups of the two letter code is otherwise identical to the three letter.
  • Table C2 provides a translation of the two or three letter codes to their chemical names.
  • An x between two nucleotides represents a phosphorothioate intemucleoside linkage.
  • An o between two nucleotides represents a phosphodiester linkage.
  • Table C3 shows selected compounds of the present invention in HELM Annotation Format.
  • the antisense oligonucleotide is a compound as shown in Table C3.
  • Table C3 Exemplary compounds of the present invention - HELM Annotation Format
  • [LR](G) is a beta-D-oxy-LNA guanine nucleoside
  • [LR](T) is a beta-D-oxy-LNA thymine nucleoside
  • [LR](A) is a beta-D-oxy-LNA adenine nucleoside
  • [LR]([5meC]) is a beta-D-oxy-LNA 5-methyl cytosine nucleoside
  • [MOE](G) is a 2'-O-Methoxy ethyl guanine nucleoside
  • [MOE](T) is a 2'-O-Methoxy ethyl thymine nucleoside
  • [MOE](A) is a 2'-O-Methoxy ethyl adenine nucleoside
  • [MOE]([5meC]) is a 2'-O-Methoxyethyl-5-methyl cytosine nucleoside
  • [mR](G) is a 2'-O-methyl RNA guanine nucleoside
  • [mR](U) is a 2'-O-methyl RNA uracil nucleoside
  • [mR](A) is a 2'-O-methyl RNA adenine nucleoside
  • [mR](C) is a 2'-O-methyl RNA cytosine nucleoside
  • [dR](G) is a DNA guanine nucleoside
  • [dR](T) is a DNA thymine nucleoside
  • [dR](A) is a DNA adenine nucleoside
  • [dR](C) is a DNA cytosine nucleoside
  • [dR]([5meC]) is a DNA 5-methyl cytosine nucleoside
  • [R](G) is a RNA guanine nucleoside
  • [R](T) is a RNA thymine nucleoside
  • [R](A) is a RNA adenine nucleoside
  • [R](C) is a RNA cytosine nucleoside
  • [sP] is a phosphorothioate internucleoside linkage
  • P is a phosphate internucleoside linkage
  • ASO ID 66 117 binds to a target sequence with Target ID No 66.
  • the compound might also bind to other target IDs of the present invention (as some target IDs overlap, see e.g. Table DI).
  • the compound selected from Table C 1 is a compound which resulted in an efficient downregulation of the target gene in the studies described in Example 5. The results for the experiments are shown in Table 8.
  • the compound is selected from the compounds resulting in an expression level of NAT8L of 50% or less than 50% in relation to PBS treated control cells.
  • the compound is selected from the compounds resulting in an expression level of NAT8L of 40% or less than 40% in relation to PBS treated control cells.
  • the antisense oligonucleotide (or composition) of the present invention shall be administered to the CNS, in particular to the brain.
  • the ASO is delivered to CNS through intrathecal injection - as it is e.g. the current state of art for similar ASO e.g. Nusinersen/Spinraza.
  • the ASO of the present invention or the pharmaceutical composition is, preferably, administered intrathecally.
  • the ASO can be administered by subcutaneous or intravenous administration either with or with or without a conjugate in order to reach the peripheral nervous system, which is also negatively affected by the loss of ASPA expression.
  • the present invention further relates to a conjugate comprising the antisense oligonucleotide of the present invention and a conjugate moiety.
  • the conjugate moiety is covalently bound to the antisense oligonucleotide, e.g. via one or more linker nucleotides, such as one, two, three or four linker nucleotides (or more).
  • the linker may be cleaved after administration to the patient.
  • the antisense oligonucleotide of the present invention shall be delivered or administered to the CNS, in particular to the brain.
  • the conjugate moiety is a moiety that allows the crossing of the conjugate of the blood brain barrier.
  • the moiety can be and antibody or antigen-binding fragment thereof targeting the transferrin receptor.
  • the antisense oligonucleotides of the present invention can be administered/delivered ‘unassisted’ in saline solution. However, distribution to certain tissues and uptake in cells can be enhanced by conjugates and formulation techniques. Conjugation to ASOs could be, peptides, antibodies and aptamers binding to receptors on target cells or proteins mediating transcytosis e.g. the transferrin receptor. Antisense oligos can also be conjugated to naturally occurring ligands or modifications hereof as exemplified by GalNac conjugation binding with high affinity to asialoglycoprotein receptor 1 (ASGR1, ASPGR) and Alpha-tocopherol conjugation and interaction with transfer protein Alfa-TTP.
  • ASOs conjugation to ASOs
  • ASOs could be, peptides, antibodies and aptamers binding to receptors on target cells or proteins mediating transcytosis e.g. the transferrin receptor.
  • Antisense oligos can also be conjugated to naturally occurring
  • tissues delivery and cellular uptake of ASOs of the present invention can be enhanced through formulation with nanocarriers, that facilitates crossing of biological barriers such as cellular membranes.
  • nanocarriers Various types of nanocarriers have been described with with favorable properties for delivery of nucleic acids e.g. lipid nanoparticles (LNPs) as used for BioNTech mRNA vaccines, LNPs functionalizes with peptides, pegylated lipids, cationic lipids, exomes (lipid bilayers) both artificial and natural exosomes such as milk exosomes and spherical nucleic acids and others as described in further details in Roberts et al., 2020 (Thomas C Roberts, Robert Langer, Matthew J A Wood. Review Nat Rev Drug Discov. 2020 Oct;19(10):673-694. Advances in oligonucleotide drug delivery).
  • the present invention further relates to a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or the conjugate of the present invention.
  • a pharmaceutical composition comprises the antisense oligonucleotide or the conjugate of the present invention together with a pharmaceutically acceptable carrier and/or, in particular, a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable refers to the non-toxicity of a material which, in certain exemplary embodiments, does not interact with the action of the oligonucleotide or the conjugate present in the pharmaceutical composition.
  • carrier refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application.
  • excipient is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surface active agents, preservatives, emulsifiers or buffer substances.
  • binders e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol
  • fillers e.g., lubricants, thickeners, surface active agents, preservatives, emulsifiers or buffer substances.
  • the form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc.
  • the pharmaceutical composition can be formulated for intrathecal administration.
  • the antisense oligonucleotide or conjugate of the present invention is preferably administered by intrathecal administration a route of administration for drugs via an injection into the spinal canal. Thereby, it reaches the cerebrospinal fluid and the brain.
  • the present invention further relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in medicine.
  • the present invention relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use treating Canavan disease.
  • the present invention relates to the use of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament for treating Canavan disease.
  • Canavan disease also referred to as Canavan-van Bogaert-Bertrand disease is a degenerative disease that is associated with a progressive damage to nerve cells and loss of white matter in the brain. The disease is inherited in an autosomal recessive manner. It is caused by mutations the ASPA gene which codes for the enzyme aspartoacylase. Decreased aspartoacylase activity prevents the normal breakdown of N-acetyl aspartate.
  • the present invention relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use treating Canavan disease.
  • the present invention relates to the use of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament for treating Canavan disease. Further, the present invention relates to a method of treating Canavan disease, comprising administering pharmaceutically effective amount of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention to a subject suffering from Canavan disease.
  • treating refers to the administration of a compound or composition or a combination of compounds or compositions to a subject in order to: ameliorate Canavan disease.
  • the term encompasses both the amelioration of one or more symptoms of the Canavan disease or prevention of the worsening of one or more symptoms, i.e. prophylaxis.
  • the amelioration of symptoms also includes the reduction of one or more symptoms.
  • the term preferably, refers to the reduction of one or more symptoms of the disease.
  • the treatment is typically a disease modifying treatment that reduces one or more symptoms of Canavan disease.
  • the development of disease pathology is inhibited. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic or disease modifying. It is to be understood that the treatment does not allow a complete cure of Canavan disease.
  • Treatment also includes increasing the life expectancy of a subject (as compared to an untreated subject).
  • the subject to be treated is a subject suffering from Canavan disease.
  • the patient might not yet show symptoms of Canavan disease at the time of the treatment.
  • the subject shows symptoms of Canavan disease.
  • Symptoms of Canavan disease are well known in the art and include (but are not limited to) one or more of lack of motor development, macrocephaly, lack of head control and abnormal muscle tone.
  • the subject has been diagnosed through genetic testing to suffer from Canavan disease.
  • the disease is diagnosed at the infant age.
  • the diagnosis involves genetic testing and/or the detection of increased levels of N-acetylaspartic acid (NAA) in the urine.
  • NAA N-acetylaspartic acid
  • the terms “subject” and “patient” are used interchangeably herein.
  • the “subject” or “patient” may be a vertebrate.
  • the term includes both humans and other animals, particularly mammals, and other organisms.
  • the subject is a mammal.
  • the subject is a primate.
  • the subject is a human subject suffering from Canavan disease.
  • the present invention further relates to a method for identifying a candidate compound for the treatment of Canavan disease, comprising a) providing an antisense oligonucleotide according to the present invention, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
  • the antisense oligonucleotide is preferably the antisense oligonucleotide of the present invention.
  • it shall comprise a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in the human NAT8Lgene (i.e. mRNA or premRNA).
  • a target sequence in the human NAT8Lgene i.e. mRNA or premRNA.
  • the definitions provided herein above preferably apply mutatis mutandis.
  • said method is an in vitro method.
  • step a) of the above method of the present invention an antisense oligonucleotide of the present invention is provided.
  • the antisense oligonucleotides are complementary to a target region as set forth herein elsewhere.
  • step b) the antisense oligonucleotide shall be contacted with a host cell. Said host cell shall express the NAT8L gene.
  • An antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in the human NAT8L (N-acetyltransferase 8 like) gene.
  • antisense oligonucleotide of any one of embodiments 1 to 4 comprising a stretch of at least 12, or at least 14 nucleotides which is at least 90% complementary to said target sequence.
  • the antisense oligonucleotide embodiment 10 wherein the target cell is a human cell, such as a cell of the CNS.
  • the antisense oligonucleotide of any one of embodiments 11 to 14, wherein the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2’-O-Methoxyethyl-RNA, 2’-O-Methyl-RNA, 2’-Fluoro-RNA.
  • modified intemucleoside linkage such as at least one Phosphorothioate intemucleoside linkage, at least one Phosphorodithioate intemucleoside linkage, at least one Phophoroamidate intemucleoside linkage, at least one methyl phospho
  • a pharmaceutical composition comprising the antisense oligonucleotide of any one of embodiments 1 to 23 or the conjugate of embodiment 24.
  • a method for identifying a candidate compound for the treatment of Canavan disease comprising a) providing an antisense oligonucleotide as defined in any one of embodiments 1 to 23, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
  • Oligonucleotide synthesis is generally known in the art and can be acquired from multiple different providers. In the listed in vitro experiments (example 1, 2, 3, 5, 6 and 7) all oligonucleotides were acquired from Biosearch Technologies (Lystrup, Denmark). In brief, after cleavage from the solid support the oligonucleotides are cartridge purified using Ammonium acetate; Dissolved to 750pM in PBS and purity determined by LC/MS (> 80%).
  • oligonucleotides were acquired from WuXi AppTec (China). After cleavage from the solid support the oligonucleotides were HPLC-purified to >90% purity (LC-TOF/MS) and lyophylized as sodium salt followed by formulation in sterile 0,9 % sodiumchloride. Endotoxin test were performed (Kinetic turbidimetric or chromogenic LAL assay) to ensure endotoxin levels ⁇ 1.0 EU/mg.
  • Example 1 The produced compounds are shown in Table Al and Table Cl (see Figures 1 and 2). All compounds shown in Table Al were tested in A549 cells (see Example 1) and/or in HEK293 cells (see Example 2 and example 4). All compounds shown in Table Cl were tested in HEK293 cells (see Example 5). Some of the ASO compounds which led to an efficient downregulation of NAT8L were further tested for potency in HEK cells and iPSC derived neurons as well as for in vivo acute tolerability in mice and for pharmacodynamics and tolerability in non-human primates, Example 1: Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in A549 cells at single test concentration.
  • a cell-based screening assay was developed using cells with an endogenous expression of NAT8L premRNA. In that optimization process various cell densities and compound incubation periods and concentrations were tested before reaching the assay conditions as described below.
  • the A549 cells were maintained and expanded as recommended by the supplier (ECACC, acquired from Merck, 86012804-1VL). The cells were grown to 70-80% confluency, the cells are then trypsinized and resuspended in growth media. Viable cells are counted using trypan blue and a Countess 3 automatic cell counter. The appropriate number of cells are diluted in complete growth media, mixed by gentle pipetting, added to reagent reservoirs, and distributed into 96-well plates using a multichannel pipette in a total volume of 195 pl/well.
  • the diluted and heat shocked RNA is used as input template for the qPCR, using qScriptTM XLT One-Step RT-qPCR ToughMix® (cat# 95134-500) from QuantaBio and qPCR assays from Integrated DNA technologies (IDT) listed in Table 2.
  • Table 2 qPCR assay used for screening NAT8L targeting ASOs.
  • the QPCR reaction was run in 384 wells using a QuantStudio 7 Flex (applied biosystems by Thermo Fisher Scientific. Quantities of NAT8L mRNA was calculated applying the ddCT method and using the median of all the PBS treated wells within the same plate as the ‘untreated control’.
  • Example 2 Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in HEK cells at single test concentration.
  • the HEK293 cells were maintained and expanded as recommended by the supplier. The cells were grown to 70-80% confluency, the cells are then trypsinized and resuspended in growth media. Viable cells are counted using trypan blue and Vi-CELL automatic cell counter (Beckman Coulter). The appropriate number of cells are diluted in complete growth media, mixed by gentle pipetting, added to reagent reservoirs, and distributed into 96-well plates using a multichannel pipette in a total volume of 190 pl/well. Sterile PBS is added to the moats of the 96-well culture plates (NuncTM EdgeTM 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplates) to reduce evaporation and potential plate effect.
  • each plate also included 10 PBS controls (10 pL), 4 NAT8L positive control ASOs and two ASO controls targeting the ATXN3 gene.
  • 10 PBS controls (10 pL)
  • 4 NAT8L positive control ASOs and two ASO controls targeting the ATXN3 gene.
  • cells were harvested by gently aspirating and RNA was extracted using the Macher ey-Nagel NucleoSpin 96 RNA Kit, according to the manufactures instructions and eluted in 75 pl of water.
  • qPCR experiments were carried out as described in Example 1. Quantities of NAT8L mRNA was calculated applying the ddCT method and using the median of all the PBS treated wells within the same plate as the ‘untreated control’.
  • the expression level of NAT8L following ASO treatment (Table 6) is thus shown as percent of the PBS-treated wells.
  • Example 3 Determination of IC50 values of NAT8L targeting ASOs in HEK cells
  • Example 4 Overview on identified target regions within the NAT8L pre-mRNA sequence (SEQ ID NO: 1) which allow for efficiently downregulating NAT8L Target sequences that were found in the studies underlying the present invention are shown in Table Bl.
  • Targeting sequences as shown in Table B2 allowed for a very efficient down-regulation of the target gene.
  • Table B2 Target sequences
  • Example 5 Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in HEK cells
  • able 8 The table shows the expression level of NAT8L relative to untreated control (UTC%).
  • the header of each column refers to the ell line (e.g. HEK293, A549), then the experiment number of each independent experiment (e.g. 1, 2 and 3 and 4), and the concentration of
  • Example 6 Determination of IC50 values of NAT8L targeting ASOs in HEK cells To validate the hits identified in single concentration screens (Example 5) and to rank compounds on their potency a concentration response experiments were subsequently carried out for compounds showing high level of knock down.
  • Example 7 Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in iPSC derived human neurons at multiple test concentration.
  • a set of ASOs selected to cover most of the preferred regions were tested in iPSC derived human neurons to confirm activity and potency of the most active ASOs in a relevant cellular model.
  • iPSC derived neurons were maintained as recommended by the supplier (Fuji film, 01279).
  • 96-well cell culture plates (NuncTM EdgeTM 96-Well, Nunclon Delta-Treated, Flat- Bottom Microplates) were coated with sterile 0,07% polyethylenimide for 1 hour, washed with PBS and water and coated with 0,01 mg/ml of laminin for 1 hour. The laminin solution was removed with no further washing of the culture plates and iPSC derived neurons were thawed and seated at a density of 80.000 neurons pr well as described in Fuji films: iCell GlutaNeurons User's Guide in a total volume of 190pl/well plates using a multichannel pipette.
  • Table 10 Information on iPSC derived neurons
  • RNA was extracted using the RNeasy 96, QIAcube HT kit (Qiagen #74171) according to the manufactures instructions and eluted in 75 pl of water. qPCR experiments were carried out as described in Example 1. Quantities of NAT8L mRNA was calculated applying the ddCT method and using the median of all the PBS treated wells within the same plate as the ‘untreated control’. The expression level of NAT8L following ASO treatment (Table 11) is thus shown as percent of the PBS-treated wells.
  • Table 11 Expression levels of human NAT8L in iPSC derived neurons shown as %UnTreatedControl (%UTC) and as IC50 Value
  • Example 8 In vivo acute toxicity in mice of ASOs targeting NAT8L.
  • mice In vivo acute tolerability of the antisense oligonucleotides were tested in mice, with 6 mice per ASO group. Mice at 8-10 weeks of age aged were housed in European IVC cages type IIL with TAPVEI aspen bedding (Tapvei Eatonis Oil, Estonia). The cages were enriched with nesting material, wooden sticks and hiding material. The light cycle was 12-hour dark and 12-hour light. Diet was pelleted complete diet (Altromin 1324, Brogaarden), and municipal drinking water. Diet and water were administered ad libitum.
  • mice Pre-dosing analgesia was given at least 30 min before dosing is initiated, all animals received preventive pain treatment with Meloxicam (2 mg/kg SC). The mice were be anesthetized with isoflurane before dosing. For injection, the G23 needle was mounted on a stand so it precisely penetrated 3.9 mm through the mouse's skull. The dose volume of 5 pL was injected over 30 seconds and the animal was then placed back in its cage and clinically observed according to the instructions below.
  • Table 12 Average neurob ehavi oral scoring at 1 hour post dose for a 50 pg dose or 100 pg dose is summarized in table 13. If the mice had to be euthanized before 1 hour post dosing, they got a score of 20.
  • mice The generated acute toxicity data in mice shows that the observed acute toxicity is highly dependent on the ASO sequence.
  • Some ASOs e.g. 29 138 and 34 46 are highly neurotoxic and giving rise to toxic effects of a severity requiring the mice to be euthanized shortly after dosing.
  • ASOs such as 29_79, 29_84, 29_85, 29_124 and 66_36 showed only minor short lasting neurological signs.
  • ASO ID 29 85 Single dose in vivo efficacy test in African green monkeys (Chlorocebus sabaeus) To evaluate the efficacy of one of the most preferred ASOs; ASO ID 29 85 in vivo using a relevant dosing paradigm and in a species of high translational value, a single dose of ASO was delivered to African green monkeys via an IT-catheter followed by a two week in life phase before harvest of brain tissues.
  • monkeys After a 4-week recovery from IT CSF access port implantation, monkeys were fasted overnight, sedated with ketamine/xylazine and placed in a prone position. 0,5 ml of ASO or saline were injected followed by a saline flush of 0.5 mL/kg monkey.
  • monkeys were sedated intramuscularly with ketamine and xylazine to effect and euthanized with sodium pentobarbital.
  • the following brain tissues was collected with a 2 mm punch into DNase/RNase free cryotubes and snap-frozen: Frontal Cortex, Parietal Cortex, Occipital Cortex, Ventral Striatum (nucleus accumbens area), Hippocampus, Ventral Pons, Medulla, Cerebellum, Ventral Midbrain, Cervical Spinal Cord, Thoracic Spinal Cord, Lumbar Spinal Cord.
  • Brain tissue pieces were homogenized in RLT buffer using the Precellus system, then Trizol/choroform extracted and RNA from the aqueous phases were purified on a QIAcube HT using the RNeasy 96, QIAcube HT kit (Qiagen #74171).
  • qPCR was performed as described in example 1 using qPCR assays specific for monkey NAT8L and monkey UBE2D2 for normalization. The reduction is presented as relative reduction compared to the median for saline treated NHPs with 2 punctures from each brain region.
  • a single 12 mg dose of ASO ID 29 85 administered intrathecally shows significant reduction of NAT8L relative to UBE2D2 in various brain regions as seen in table 14:
  • Table 14 %NAT8L mRNA levels (Normalized to UBE2D2) of saline treated NHP "average ⁇ stdev).
  • Example 10 Overview on target regions within the NAT8L pre-mRNA sequence (SEQ ID NO: 1) identified in Example 5.
  • Target sequences of the invention that were found in Example 5 are shown in Table DI. Targeting these regions allows for an efficient downregulation of the NAT8L gene. Some of the identified target sequences, in principle, correspond to some of the target sequences described in Table Bl and are subregions or expansions of these target sequences. For more details, see the Table in the specification. Table DI: Target sequences
  • Targeting sequences as shown in Table D2 allowed for a very efficient down-regulation of the target gene.
  • Table D2 Target sequences The best results were obtained when ASOs were used that are complementary to target sequences shown in Table D3.
  • Table D4 shows target sequences of preferred compounds.

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Abstract

The present invention relates to antisense oligonucleotides (oligomers) complementary to NAT8L pre-mRNA and mRNA and which are capable of inhibiting the expression of NAT8L. Inhibition of NAT8L expression is beneficial for the treatment of Canavan disease.

Description

Antisense oligonucleotides for the treatment of Canavan disease
Field of the invention
The present invention relates to antisense oligonucleotides (oligomers) complementary to NAT8L (N-acetyltransferase 8 like) pre-mRNA and mRNA and which are capable of inhibiting the expression of NAT8L. Inhibition of NAT8L expression is beneficial for the treatment of Canavan disease.
Background
Canavan disease is a recessively inherited vacuolar leukodystrophy caused by ASPA mutations. ASPA encodes aspartoacylase, an oligodendroglial enzyme required for cleavage of the abundant brain amino acid N-acetyl-l-aspartate (NAA) to acetate and L-aspartate.
Canavan disease is a rare and fatal autosomal recessive degenerative disorder that causes progressive damage to nerve cells and pathology of the white matter in the brain. It is one of the most frequent degenerative brain diseases of infancy. Canavan disease belongs to a group of genetic diseases known as leukodystrophies, referring to defects in the development and growth of white matter. Early symptoms of Canavan disease such a hypotonia, macrocephaly, poor head control usually become apparent between the age of 3 and 6 months. The affected infants show delayed developmental milestones (e.g., sitting, standing) and usually never develop the ability to walk. The progressive nature of the disease results in loss of previously acquired abilities and additional symptoms such as seizures and mental retardation also become apparent during infancy. The prognosis for Canavan disease is poor and, in most cases, death occurs before the age of ten.
Canavan disease is caused by mutations in the in the ASPA gene, which encodes aspartoacyclase, an enzyme required conversion of the N-acetyl-l-aspartate (NAA) into acetate and L-aspartate. Currently, there are two prevailing hypotheses of the resulting pathological process. The ‘Oligodendroglial starvation’ hypothesis proposes that the lack of functional aspartoacyclase results in a deficiency of NAA-derived acetate required for the biosynthesis of myelin (white matter material) and as a result degeneration of myelin/white matter in patients. The ‘NAA toxicity’ hypothesis in contrary proposes, that the excessive NAA concentration in the brain of patients lacking aspartoacyclase, causes an impaired osmolar homeostasis and as result the astroglial and myelinic vacuolation, which is hallmark of Canavan disease (Pleasure et al., Neurochem Res. 2020 Mar;45(3):561-565. doi: 10. 1007/sl 1064-018-2693-6. Epub 2018 Dec 8. Pathophysiology and Treatment of Canavan Disease.).
In support of the ‘NAA toxicity’ Maier et al., 2015 reported that in Aspa deficient mice (Asap nur7/nur7’ Canavan disease model) also deficient for NAA synthase Nat81 (Aspanur7/nur7/ Nat81-/ ) showed normal myelin content and no axonal degeneration, despite having no detectable NAA. However, survival time was not improved (Maier et al., J Neurosci. 2015 Oct 28;35(43): 14501-16. doi: 10.1523/JNEUROSCI.1056-15.2015. N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time). Moreover, mice with only one intact Nat81 allele (Aspanur7/nur7/ Nat81 /+) accumulated less NAA and developed a less severe disease pathology showed phenotypic improvements and normal lifespan. Similar results have been reported by Sohn et al. (J Neurosci. 2017 Jan 11;37(2):413-421. doi: 10.1523/JNEUROSCI.2013-16.2016. Suppressing N-Acetyl-1- Aspartate Synthesis Prevents Loss of Neurons in a Murine Model of Canavan Leukodystrophy).
Subsequently, Bannerman et al., 2018 showed that knocking down Nat81 expression in Aspanur7/nur7 using adeno-associated viral vector carrying short hairpin Nat81 inhibitory RNA reduced NAA levels, suppressed the development disease pathology, and improved enhanced motor performance (Bannerman et al., Brain Nat81 Knockdown Suppresses Spongiform Leukodystrophy in an Aspartoacylase-Deficient Canavan Disease Mouse Model Mol Ther. 2018 Mar 7;26(3):793-800. doi: 10.1016/j.ymthe.2018.01.002. Epub 2018 Jan 10). furthermore, Hull et al., 2020 showed that antisense oligonucleotides reducing Nat81 expression in Aspanur7/nur7 likewise reduced levels of NAA, reversed ataxia and reduced the cerebellar and thalamic vacuolation in young-adult Aspanur7/nur7 mice (Ann Neurol. 2020 Mar;87(3):480-485. doi: 10.1002/ana.25674. Epub 2020 Jan 22. Antisense Oligonucleotide Reverses Leukodystrophy in Canavan Disease Mice).
Altogether these data points towards inhibition NAT8L and lowering of NAA levels as a potentially effective therapeutic concept for treating Canavan disease, a progressive and fatal neurodegenerative disease for which there are currently no effective treatments available.
There remains a need for means and methods for the efficient down-regulation of the NAT8L gene.
Brief summary of the present invention
The present invention provides an antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in a NAT8L (N- acetyltransferase 8 like) gene.
Preferably, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of the target sequences shown in Table B 1 or DI in the Examples section.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 81 and SEQ ID NO: 882 to SEQ ID NO: 906. In a preferred embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 30 to SEQ ID NO: 81. The sequences are shown in Table Bl in the Examples section.
In a further preferred embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 11 to SEQ ID NO: 29. The sequences are shown in Table B2 in the Examples section.
In another preferred embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 883, 892, 894, 895, 896, 897, 898 or 899. The sequences are shown in Table D2 in the Examples section.
In a particularly preferred embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 10. The sequences are shown in Table B3 in the Examples section.
In another particularly preferred embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 883, 894, 895, 897, 898 and 899. The sequences are shown in Table D3 in the Examples section.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 4.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 5.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 6.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 7.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 8.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 9.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 10.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 883.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 894.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 895. In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 897.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 898.
In an embodiment, the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence as shown in SEQ ID NO: 899.
Preferably, the antisense oligonucleotide comprises a stretch of at least 12, or more preferably at least 14 nucleotides which is at least 90% complementary to a target sequence as set forth herein.
Also preferable, the target sequence comprises or consists of a nucleic acid sequence as shown in a sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 402 and SEQ ID NO: 907 to SEQ ID NO: 936.
In preferred embodiment, the stretch is 95%, in particular 100% complementary to the target sequence.
Preferably, the antisense oligonucleotide has a length of 12 to 30 nucleotides, more preferably, a length of 14 to 22 nucleotides, and most preferably a length of 16 to 20 nucleotides.
In some embodiments, the antisense oligonucleotide comprises or consists of a nucleic acid sequence as shown in SEQ ID NO: 403 to SEQ ID NO: 881, such as in SEQ ID NO: 403 to SEQ ID NO: 723, or in SEQ ID NO: 724 to SEQ ID NO: 881.
In some embodiments, the antisense oligonucleotide is an antisense compound as shown in Table Al in Figure 1, wherein
Adx represents 2'deoxyadenosine-3' -phosphorothioate
Aox represents 2'-O-methyladenosine-3'-phosphorothioate
Amx represents 2'-O-Methoxyethyladenosine-3' -phosphorothioate
Alx represents 2'-O-beta-D-oxy LNA adenosine-3' -phosphorothioate
Cdx represents 2'deoxycytidine-3' -phosphorothioate
Cox represents 2'-O-methylcytidine-3 '-phosphorothioate
Cmx represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphorothioate
Clx represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphorothioate
Edx represents 2'deoxy-5-methylcytidine-3' -phosphorothioate
Emx represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphorothioate
Elx represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphorothioate
Gdx represents 2'deoxyguanosine-3' -phosphorothioate
Gox represents 2'-O-methylguanosine-3 '-phosphorothioate
Gmx represents 2'-O-Methoxyethylguanosine-3' -phosphorothioate
Glx represents 2'-O-beta-D-oxy LNA guanosine-3' -phosphorothioate Tdx represents 2'deoxythymidine-3' -phosphorothioate
Tmx represents 2'-O-Methoxyethylthymidine-3' -phosphorothioate
Tlx represents 2'-O-beta-D-oxy LNA thymidine-3' -phosphorothioate
Uox represents 2'-O-methyluridine-3'-phosphorothioate
Ado represents 2'deoxyadenosine-3' -phosphate
Aoo represents 2'-O-methyladenosine-3 '-phosphate
Amo represents 2'-O-Methoxyethyladenosine-3' -phosphate
Alo represents 2'-O-beta-D-oxy LNA adenosine-3' -phosphate
Cdo represents 2'deoxycytidine-3' -phosphate
Coo represents 2'-O-methylcytidine-3 '-phosphate
Cmo represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphate
Elo represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphate
Edo represents 2'deoxy-5-methylcytidine-3' -phosphate
Emo represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphate
Elo represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphate
Gdo represents 2'deoxyguanosine-3' -phosphate
Goo represents 2'-O-methylguanosine-3'-phosphate
Gmo represents 2'-O-Methoxyethylguanosine-3' -phosphate
Gio represents 2'-O-beta-D-oxy LNA guanosine-3' -phosphate
Tdo represents 2'deoxythymidine-3' -phosphate
Tmo represents 2'-O-Methoxyethylthymidine-3' -phosphate
Tlo represents 2'-O-beta-D-oxy LNA thymidine-3' -phosphate
Uoo represents 2'-O-methyluridine-3'-phosphate
Ad represents 2'deoxyadenosine-3'
Ao represents 2'-O-methyladenosine-3'
Am represents 2'-O-Methoxyethyladenosine-3'
Al represents 2'-O-beta-D-oxy LNA adenosine-3'
Cd represents 2'deoxycytidine-3'
Co represents 2'-O-methylcytidine-3'
Ed represents 2'deoxy-5-methylcytidine-3'
Em represents 2'-O-Methoxyethyl-5-methylcytidine-3'
El represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3'
Gd represents 2'deoxyguanosine-3'
Go represents 2'-O-methylguanosine-3'
Gm represents 2'-O-Methoxyethylguanosine-3'
G1 represents 2'-O-beta-D-oxy LNA guanosine-3'
Td represents 2'deoxythymidine-3'
Tm represents 2'-O-Methoxyethylthymidine-3',
T1 represents 2'-O-beta-D-oxy LNA thymidine-3', and
Uo represents 2'-O-methyluridine-3'. In some embodiments, the antisense oligonucleotide is an antisense compound as shown in Table Cl in Figure 2.
In an embodiment, the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2 17, 2_35, 2_50, 2_51, 2_52, 6_8, 25 113, 29_2, 29_5, 29_6, 29_7, 29_10, 29_23, 29_24, 29_32, 29_34, 29_36, 29_54, 29_70, 29_72, 29_78, 29_79, 29_84, 29_85, 29_86, 29_100, 29_112, 29_121, 29_123, 29_124, 29_125, 29_130, 29_133, 29_138, 29_154, 34_38, 34_39, 34_46, 34_47, 37 15, 38 5, 38 10, 38 11, or 51 66 as shown in Table Al. The ASO compounds with these ASO IDs had the best effect on the down-regulation of the target gene (see Examples 1 and 2).
In preferred embodiment, the antisense oligonucleotide is an antisense oligonucleotide compound selected from the compounds shown in Table A3. Here, the compounds are provided in the so called HELM annotation format.
In another preferred embodiment, the antisense oligonucleotide is an antisense oligonucleotide compound selected from the compounds shown in Table C3. Here, the compounds are provided in the so called HELM annotation format. Thus, the antisense oligonucleotide is an preferably antisense oligonucleotide with ASO ID 2_17, 25 111, 25_113, 29_10, 29_124, 29_130, 29_138, 29_24, 29_34, 29_36, 29_5, 29_70, 29_78, 29_79, 29_84, 29_85, 29_86, 34_39, 34_46, 37 15, 38 10, 51 6, 66_117, 66_120, 66_134, 66_135, 66_123, 66_124, 66_130, 66_126, 66_127, 66_149, 66_189, 66_181, 66_182, 66_173, 66_153, 66_160, 66_185, 66_183, 66_177, 66_188, 66_140, 66_176, 66_174, 66_217, 66_220, 66_27, 66_36, 66_39, 66_408, 66_412, 66_430, 66_431, 66_456, 66_459, 66_47, 66_485, 66_492, 66_496, 66_545, 66_547, 66_567, 66_573, 66_584, 66_587, 66_576, 66_588, 66_63, 66_592, 66_593, 66_600, 66_64, 68_16, 54_1, 54_3, 54_2 or 66_544.
More preferably, the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2_17, 25 113, 29_10, 29_124, 29_70, 29_78, 29_79, 29_84, 29_85, 29_86, 34_39, 51_6, 66_120, 66_134, 66_135, 66_124, , 66_130, 66_126, 66_127, 66_149, 66_181, 66_182, 66_173, 66_160, 66_185, 66_183, 66_177, 66_188, 66_140, 66_176, 66_174, 66_220, 66_27, 66_36, 66_39, 66_408, 66_430, 66 431, 66_456, 66_47, 66_485, 66_492, 66_496, 66_545, 66_547, 66_567, 66_573, 66_584, 66_587, 66_576, 66_588, 66_63, 66_592, 66_593, 66_600, 66_64, 54_3, 54_2, or 66_544.
Most preferably, the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2_17, 29_124, 29_79, 29_84, 29_85, 29_86, 66_134, 66_135, 66_124, 66_130, 66_126, 66_149, 66_181, 66_182, 66_173, 66_160, 66_185, 66_183, 66_177, 66_188, 66_140, 66_176, 66_174, 66_220, 66_27, 66_36, 66_39, 66_408, 66_47, 66_485, 66_496, 66_545, 66_547, 66_567, 66_573, 66_584, 66_587, 66_576, 66_588, 66_63, 66_592, 66_593, 66_600, 66_64, 54_3, 54_2 or 66_544.
The antisense oligonucleotide of the present invention shall be capable of reducing the amount of NAT8L (N-acetyltransferase 8 like) mRNA in a host cell expressing said NAT8L mRNA. Typically, the host cell is a mammalian cell, such as a primate cell. In a preferred embodiment, said host cell is human host cell.
Preferably, the antisense oligonucleotide is a chemically modified antisense oligonucleotide. A chemically modified antisense oligonucleotide typically comprises modifications of the phosphodiester backbone chemistry, nucleobase modifications and sugar modifications.
In a preferred embodiment, the chemically modified antisense oligonucleotide comprises at least one 2’ modified sugar or bicyclic sugar.
Alternatively or additionally, the chemically modified antisense oligonucleotide contains at least one modified nucleobase. For example, at least one modified nucleobase is 5 -methylcytosine.
Alternatively or additionally, the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2'-O-Methoxyethyl-RNA, 2’-O- Methyl-RNA, 2’-Fluoro-RNA.
Further, the chemically modified antisense oligonucleotide may comprise at least one modified intemucleoside linkage. In an embodiment, at least five, such as at least 10 intemucleoside linkages are modified intemucleoside linkages. In an embodiment, all intemucleoside linkages are modified intemucleoside linkages.
A “modified intemucleoside linkage” as used herein, refers to an intemucleoside linkage other than a phosphodiester linkage. Thus, the antisense oligonucleotide may comprise unmodified intemucleoside linkages (i.e. phosphodiester linkages), modified intemucleoside linkages, or a combination thereof.
Preferably, the modified linkage(s) is (are) selected from: a Phosphorothioate intemucleoside linkage, a Phosphorodithioate intemucleoside linkage, a Phophoroamidate intemucleoside linkage, a methyl phosphonate intemucleoside linkage, a phosphotriester intemucleoside linkage, a boranophosphate intemucleoside linkage and a phosphoryl guanidine intemucleoside linkage. Moreover, intemucleoside linkage can be stereodefmed versions of said linkages.
More preferably, the at least one modified linkage is a phosphorothioate linkage. Most preferably, at least 50% of the intemucloside linkages, such as all intemucleoside linkages, are phosphorothioate intemucleoside linkages.
Preferably, the antisense oligonucleotide comprises at least one nucleoside with a modified sugar moieity, typically at least four nucleosides with a modified sugar moiety, (herein also referred to as sugar modified nucleosides). In an embodiment, the antisense oligonucleotide comprises at least one, such as one, two, three, four or more LNA (locked nucleic acid) or MOE (2’-O-Methoxyethyl) nucleosides. For example, the LNA nucleoside is a beta-D-oxy LNA nucleoside.
Moreover, it is envisaged that the antisense oligonucleotide has a gapmer structure, i.e. is a gapmer.
The present invention further relates to a conjugate comprising the antisense oligonucleotide according to the present invention, wherein the said antisense oligonucleotide is covalently attached to a conjugate moiety.
The present invention further relates to pharmaceutical composition comprising the antisense oligonucleotide according to the present invention or the conjugate according to the present invention. In an embodiment, the composition further comprises diluents and carriers.
The present invention further relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating Canavan disease.
The present invention further relates to a method for treating Canavan disease, comprising administering to a subject suffering from Canavan disease a pharmaceutically effective amount of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in treating Canavan disease.
The present invention further relates to a method for identifying a candidate compound for the treatment of Canavan disease, comprising a) providing an antisense oligonucleotide according to the present invention, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
Figures
Figure 1: Table Al with information on antisense compounds tested in the studies underlying the present invention.
Figure 2: Table Cl with information on antisense compounds tested in the studies underlying the present invention.
Detailed overview on the present invention Inhibition of NAT8L (N-acetyltransferase 8 like) expression is considered as therapeutic concept for treating Canavan disease. In the studies underlying the present invention, target regions within the NAT8L pre-mRNA were identified which - when targeted by antisense oligonucleotides - allow for efficient downregulation of the human NAT8L pre-mRNA (or mRNA) in a host cell expressing said pre-mRNA or mRNA (see Tables 3, 6 and 8). Further, down-regulation of expression was observed in a neuronal cell line. The sequences of the target regions are shown in Tables Bl, B2, B3 and DI. Thus, the invention provides antisense oligonucleotides which are capable of downregulating NAT8L. Preferably, the antisense oligonucleotides (ASOs) comprise a stretch of at least 10 nucleotides which is preferably 90%, more preferably, 95% and most preferably fully complementary (i.e. 100% complementary) to the target region (herein also referred to as target sequence). The antisense oligonucleotides of the present invention are candidates for the treatment of Canavan disease. Advantageously, compounds with a low neuronal toxicity were identified. Moreover, after administration of a compound of the present invention to a non-human primate no adverse side effects were observed.
Accordingly, the present invention relates to an antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in a NAT8L (N- acetyltransferase 8 like) gene.
The term “oligonucleotide” as used herein is well known in the art. As used herein, the term refers to a molecule of at least ten covalently linked nucleotides. Typically, the oligonucleotides as referred to herein are chemically synthesized, for example by solid-phase chemical synthesis. The oligonucleotides as referred to herein shall contain various chemical modifications which typically do occur in nature. For example, the antisense oligonucleotide may contain at least one 2’ modified sugar. In a preferred embodiment, the antisense oligonucleotides are gapmers. The oligonucleotides of the present invention are antisense oligonucleotides, and in particular single-stranded oligonucleotides. Accordingly, they shall be capable of binding the NAT8L gene, in particular to the NAT8L pre-mRNA, when expressed in a cell, thereby down-regulating the expression of NAT8L gene in the cell. In an embodiment, the cell is a human cell is a cell of the central nervous system (CNS). Typically, the cell is a brain cell.
The NAT8L (N-acetyltransferase 8 like) gene is well known the art. The NAT8L gene is typically the human NAT8L gene. Information on the gene, such as on the nucleic acid sequence, can be found in the known databases, for example, under NCBI Gene ID: 339983). Alternative names of the gene are FLJ37478, Hcml3, Shaft or N-acetylaspartate synthetase gene)
The human NAT8L gene encodes a protein having N-acetylaspartate synthetase activity (EC 2.3.1.17). Accordingly, it catalyzes the synthesis of N-acetylaspartate acid (NAA) from L-aspartate and acetyl-CoA. The protein sequence can be assessed in the Uniprot database under the accession number Q8N9F0 (NAT8L_HUMAN). Typically, the human NAT8L protein has an amino acid sequence as shown in SEQ ID NO: 3 (which is encoded by a transcript having a sequence as shown in SEQ ID NO: 2).
The NAT8L protein is typically referred to as “N-acetyltransferase 8 like”. Alternative names are N- acetylaspartate synthetase, NAA Synthetase, or aspartate N-acetyltransferase).
Typically, the ASO of the present invention targets the human NAT8L pre-mRNA, i.e. downregulates expression of said pre-mRNA. The sequence of the human NAT8L pre-mRNA can be e.g. assessed in the Ensembl database under accession number in ENST00000423729.3. It is encoded by a region on human Chromosome 4: position 2,059,327-2,069,089 on the forward strand (Assembly GRCh38). The sequence of the human pre-mRNA has a sequence as shown in SEQ ID NO: 1. Within the cell, the pre-mRNA is further processed, i.e., by splicing, thereby generating a protein coding mRNA (herein also referred to as transcript). In some embodiments, the antisense oligonucleotide of the present invention may also target the human NAT8L mRNA (if the target region is located within an exon, either coding or in the 3’- or 5’UTR). For example, the antisense oligonucleotide of the present invention may target the human NAT8L mRNA having a sequence as shown in SEQ ID NO: 3. SEQ ID NO: 1 and 3 are RNA sequences. In the sequence listing, they are provided as DNA sequences. It is understood by the skilled person that the target RNA sequences have uracil (U) bases instead of thymidine bases (T). The same applies to the other target sequences as referred to herein (such as SEQ ID NO: 10 to SEQ ID NO: 402 or SEQ ID 882 to 936) which are provided as DNA sequences as well.
The human NAT8L pre-mRNA comprises three exons and two introns. An overview on the location of the introns, the exon, and the 3 ’ and 5 ’ UTR with the pre-mRNA sequence can be found in the following table.
Overview on UTR, exon and intron regions in the human NAT8L pre-mRNA
Figure imgf000011_0001
As set forth above, the antisense oligonucleotides of the present invention shall be capable of downregulating, i.e. reducing expression of the NAT8L mRNA in a cell that expresses said mRNA. Preferably, the expression is reduced in a call by antisense oligonucleotides of the present invention by least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a control cell (i.e. an untreated control cell). How to assess whether the expression is reduced can be assessed by well-known methods, i.e. by measuring the expression level (i.e. the amount of the target mRNA) in ASO treated cells. In an embodiment, the down-regulation of the target gene is assessed as described in the Examples section. As control for down regulation untreated cells can be used. Down-regulating the expression of the NAT8L mRNA, typically, leads downregulation of the NAT8L protein and thus to reduced levels of N-acetyl-L- Aspartate (NAA) as compared to a control. Down-regulation of the NAT8L protein can be assessed by e.g. assessing the N-acetylaspartate synthetase activity in cells treated with the ASO of the present invention by using well known enzymatic assays or by or quantifying the protein expression, such as by Western Blotting, mass spectrometry or ELISA. Preferably, the N-acetylaspartate synthetase activity is reduced in a cell by antisense oligonucleotides of the present invention by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to a control cell (i.e. an untreated control cell).
The above assessments can be done in vivo or in vitro. If they are done in vitro, they are typically done in human cells, such as in human cells used in the Examples section. In vivo, it is e.g. envisaged that a down-regulation of the target mRNA or protein, of at least 30%, such as at least 40% is achieved. For example, the down-regulation of the target mRNA or protein may be between 40% to 60%, or between 50% to 60% as compared to a control.
As set forth above, target sequences within the NAT8L pre-mRNA were identified which can be efficiently targeted with ASOs. In total, 77 of such target regions/sequences were identified. 52 of these target regions are shown in the following table. In the table, each identified target region was assigned a so called “Target ID” (Target ID 1 to 52). These IDs are used throughout the application. The terms “target region”, “target sequence” and “target nucleic acid” are used interchangeably herein.
Overview on locations of target regions identified within the NAT8L pre-mRNA sequence (Part 1)
Figure imgf000012_0001
Figure imgf000013_0001
Further information on the above target regions can be found in Table Bl in the Example 4 (e.g. the sequence of the target region or the SEQ ID NO). Tables B2 and B3 in Example 4 list preferred target regions. The target regions in Tables B2 and B3 may be present in the target regions shown in Table Bl, but may be shorter.
Further target regions are shown in the following table. Again, each identified target region was assigned a so called “Target ID” (Target ID 53 to 77). Some of the target regions shown in the following Table correspond to the target regions in the above table. For example, the target region with Target ID 53 is a subregion of the target region with Target ID 2, i.e. it is contained in this region. For more details, please see the column “Comments”. Overview on locations of target regions identified within the NAT8L pre-mRNA sequence (Part 2)
Figure imgf000014_0001
Further information on the above target regions can be found in Table DI in the Examples section (e.g. the sequence of the target region or the SEQ ID NO). Tables D2 and D3 list preferred target sequences.
Preferably, the antisense oligonucleotide of the present invention is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in the above table or in Table B 1 in Example 5. Thus, the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 30 to SEQ ID NO: 81.
More preferably, the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table B2. Accordingly, the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 11 to SEQ ID NO: 29.
Most preferably, the antisense oligonucleotide is capable of binding (i.e. hybridizing, i.e. complementary) to a target region selected from a target region shown in Table B3. Accordingly, the target sequence has a sequence selected from the sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 10.
Also preferably, the antisense oligonucleotide of the present invention is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in the above table or in Table D 1 in the Examples section. Thus, the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 882 to SEQ ID NO: 906. More preferably, the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table D2. Accordingly, the target sequence has a sequence selected from the group of target sequences consisting of SEQ ID NO: 883, 892, 894, 895, 896, 897, 898 and 899. Most preferably, the antisense oligonucleotide is capable of binding (i.e. hybridizing) to a target region selected from a target region shown in Table D3. Accordingly, the target sequence has a sequence selected from the sequences consisting of SEQ ID NO: 883, 894, 895, 897, 898 and 899.
Accordingly, the antisense oligonucleotide typically comprises stretch of at least 10 nucleotides which is at least 90% complementary (such as 95% or 100%) to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 81 and SEQ ID NO: 882 to SEQ ID NO: 906.
In a preferred embodiment, the target sequence has a sequence as shown in SEQ ID NO: 4.
In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID NO: 5. In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID NO: 6. In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 7.
In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 8. In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 9. In another preferred, embodiment, the target sequence has a sequence as shown in SEQ ID NO: 10. In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID: 894. In another preferred embodiment, the target sequence has a sequence as shown in SEQ ID: 883. In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 895.
In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 897.
In another preferred, the target sequence has a sequence as shown in SEQ ID NO: 898.
In another preferred, embodiment, the target sequence has a sequence as shown in SEQ ID NO: 899.
Further, the target sequence, preferably comprises a sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 402. Alternatively, the target sequence comprises a sequence selected from the group consisting of SEQ ID NO: 907 to SEQ ID NO: 936 (see Table D4). Also preferably, the target sequences consists of a sequence selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 402, and SEQ ID NO: 907 to SEQ ID NO: 936.
As set forth above, the antisense oligonucleotides of the present invention are preferably singlestranded antisense oligonucleotides. Preferably, the antisense oligonucleotides of the present invention are not inhibitory RNAs. In particular, the antisense oligonucleotides of the present invention are not siRNAs or short-hairpin RNAs.
In order to bind to a target sequence as referred to herein, the antisense oligonucleotides of the invention shall comprise a “stretch of nucleotides” which is sufficient complementary to a target sequence as referred to herein. In an embodiment, the stretch of nucleotides is at least 90% complementary to a target sequence. In another embodiment, the stretch of nucleotides is at least 95% complementary to a target sequence. In particular preferred embodiment, the stretch of nucleotides is fully complementary (i.e. 100% complementary to the target sequence). The term “complementary” is well known in the art. The percentage of complementary is typically calculated by calculating the proportion of nucleotides (in %) within the stretch of oligonucleotides of the ASO of the present invention which are complementary to the target sequence within the NAT8L gene. A nucleotide present in the ASO of the present invention are considered as complementary if it forms a Watson-Crick base pair with the nucleotide present in the target RNA sequence. Watson Crick base pairs are guanine-cytosine; adenine -uracil, and adenine- thymine, i.e. G-C, A-U or A-T. As will be understood by the skilled person modified nucleotides have also the capacity to form such base pairs. For more information, see e.g. Table A2.
As will be understood by the skilled person, the “stretch of nucleotides” as referred to herein needs to have a certain length in order to allow for the binding of the oligonucleotide of the present invention to the target region. Preferably, the stretch of nucleotides has a length of at least 10 nucleotides, more preferably of at least 12 nucleotides and most preferably of at least 14 nucleotides. Further, the antisense oligonucleotide of the present invention may comprise further nucleotides - i.e. in addition to the stretch of nucleotides as referred to above, such as linker nucleotides. These further nucleotides may be complementary to the target sequence, or not.
In total, the antisense oligonucleotide of the present invention, preferably, has a length of 12 to 30 nucleotides, more preferably, of 14 to 22 nucleotides, and most preferably of 16 to 20 nucleotides. Accordingly, it is envisaged that the antisense oligonucleotide in not longer than 30 nucleotides. In some embodiments, the antisense oligonucleotide in not longer than 22 nucleotides or 20 nucleotides.
In a preferred embodiment, the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 403 to SEQ ID NO: 723. In another preferred embodiment, the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 403 to SEQ ID NO: 723.
In yet another preferred embodiment, the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 724 to 881. In another embodiment, the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 724 to 881.
In a particular preferred embodiment, the antisense oligonucleotide comprises or consists of a nucleic acid sequence selected from SEQ ID NO: 724 to 881. Preferably, the antisense oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 410, 517, 558, 580, 582, 584, 661, 557, 570, 573, 613, 614, 621, 630, 707, 725, 799, 801, 802, 803, 805, 785, 787, 819, 822, 823, 789, 828, 829, 830, 831, and 835. More preferably, the antisense oligonucleotide consists of a nucleic acid sequence selected from SEQ ID NO: 410, 517, 558, 580, 582, 584, 661, 557, 570, 573, 613, 614, 621, 630, 707, 725, 799, 801, 802, 803, 805, 785, 787, 819, 822, 823, 789, 828, 829, 830, 831, and 835.
Further, it is envisaged that the antisense oligonucleotide comprises, preferably, at least 10, more preferably at least 12, even more preferably at least 14 and most preferably, at least 15 consecutive nucleotides of the sequences selected from SEQ ID NO: 403 to 881. Moreover, it is envisaged that the antisense oligonucleotide comprises, preferably, at least 10, more preferably at least 12, even more preferably at least 14 and most preferably, at least 15 consecutive nucleotides of the compounds shown in Table Al and Cl.
In an embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 410 (see e.g. compound with ASO ID 2 17).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 413 (see e.g. compound with ASO ID 2_50, 2_52, or 2_35).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 517 (see e.g. compound with ASO ID 25 111 or 25 113). In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 557 (see e.g. compound with ASO ID 29 5).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 558 (see e.g. compound with ASO ID 29 10).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 561 (see e.g. compound with ASO ID 29 34).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 570 (see e.g. compound with ASO ID 29_70 ).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 573 (see e.g. compound with ASO ID 29_78, 29_79, 29_84, 29_85 or 29_86).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 580 (see e.g. compound with ASO ID 29_124 or 29 121).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 582 (see e.g. compound with ASO ID 29_130).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 584 (see e.g. compound with ASO ID 29 131).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 613 (see e.g. compound with ASO ID 34 39).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 614 (see e.g. compound with ASO ID 34 46).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 621 (see e.g. compound with ASO ID 37 15).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 630 (see e.g. compound with ASO ID 38 10).
In another embodiment, the antisense oligonucleotide comprises or, in particular, consists of a nucleic acid sequence as shown in SEQ ID NO: 707 (see e.g. compound with ASO ID 51 6).
The sequences and compounds referred to above can be found in the Table Al in Figure 1.
Preferably, the antisense oligonucleotide of the present invention is a chemically modified antisense oligonucleotide. Accordingly, it does not occur in nature. As known by the skilled person a wide range of chemical modification can be incorporated into an oligonucleotide, such modification are e.g. reviewed in Crooke et al. which herewith is incorporated by reference in its entirety (Stanley T Crooke, Xue-Hai Liang, Brenda F Baker, Rosanne M Crooke. Review J Biol Chem. 2021 Jan- Jun;296. antisense technology: A review), which herewith is incorporated by reference in its entirety. Preferably, at least one of the nucleotides (herein also referred to a monomer) present in the oligonucleotide comprises a chemical modification. More preferably, at least 30%, such as at least 50% of the nucleotides present in the oligonucleotide comprise a chemical modification. In some embodiments, all of the nucleotides comprise chemical modification. Modifications include modifications of the phosphodiester backbone chemistry (“backbone modifications”), nucleobase modifications and sugar modifications. Preferred chemical modifications are shown in Table A2 and C2, see in particular the column “Nucleotide”. The ASOs of the present invention may comprise such nucleotides.
Modifications of the phosphodiester backbone chemistry affect the linkage between the individual monomers of the ASO. Thus, the ASO of the present preferably comprises one or more intemucleoside linkages other than a phosphodiester linkage. More preferably, the antisense oligonucleotide comprises at least five, such as at least ten modified intemucleoside linkages. Most preferably, all intemucleoside linkages are modified linkages.
Preferably, the antisense oligonucleotide comprises at least five, such as at least ten modified intemucleoside linkages, such as Phosphorothioate intemucleoside linkage. Most preferably, all intemucleoside linkages are modified linkages. However, some linkages may be phosphodiester linkages, such as one, up to two, up to three or up to four, up to six, or up to eight phosphodiester linkages.
Preferably, the at least one modified intemucleoside linkage is selected from the group consisting of: at least one Phosphorothioate intemucleoside linkage, at least one Phosphorodithioate intemucleoside linkage, at least one Phophoroamidate intemucleoside linkage, at least one methyl phosphonate intemucleoside linkage, at least one phosphotriester intemucleoside linkage, at least one boranophosphate intemucleoside linkage and at least one phosphoryl guanidine intemucleoside linkage. Moreover, the at least one modified intemucleoside linkage can be a stereodefined versions of said linkages.
In a preferred embodiment, the oligonucleotide comprises at least one phosphorodithioate intemucleoside linkage.
In another preferred embodiment, the oligonucleotide comprises at least one phosphoryl guanidine intemucleoside linkage. Most preferably, all internucleoside linkage are phosphoryl guanidine intemucleoside linkages.
In particularly preferred embodiment, the oligonucleotide comprises at least one phosphorothioate intemucleoside linkage. Most preferably, all intemucleoside linkage are phosphorothioate linkages.
Further, it is envisaged that the backbone may be modified with Morpholino Phosphorodiamidate (PMO) and Peptide Nucleic Acid (PNA).
Modification applied to the sugar group could be acyclic modifications such as UNA (unlocked nucleic acid), FNA (Flexible nucleic acid), (S)- and (R)-GNA (glycol nucleic acid), D- and L-aTNA (threofiiranosyl nucleic acids) , SNA (Serinol nucleic acids), as described in further details in Bege & Borbas 2021 (Miklos Bege & Aniko Borbas Review Pharmaceuticals (Basel) . 2022 Jul 22;15(8):909. doi: 10.3390/phl5080909. The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic).
In a preferred embodiment, the chemically modified antisense oligonucleotide comprises one or more modified nucleosides.
Preferably, the one or more modified nucleosides are sugar modified nucleosides, such as one, two, three, four or more sugar modified nucleosides. Typically, it comprises four sugar modified nucleosides. A sugar modified nucleoside is nucleoside with a modified sugar. In an embodiment, the one or more sugar modified nucleosides are 2’ sugar modified nucleosides, such as 2’0 modified sugar nucleosides.
In particular, the 2’0 modified sugar is, selected from the group consisting of 2’-0-Me, 2’MOE (2’- O-Methoxyethyl)), 2’-Npropyl, 2’-O-allyl, 2’F RNA, 2’-O-ethylamine.
In an embodiment, the 2’0 modified sugar is 2’MOE (2’-O-Methoxyethyl). Thus, the modified nucleosides are 2’MOE nucleosides.
Moreover, the one or more modified nucleotides could be locked nucleic acids such as, beta-D-oxy- LNA, 2',4'-constrained 2'-0-ethyl (cEt), such as R-cET and S-cEt, Beta-D-amino LNA, Beta-D-thio LNA, alpha-L-oxy LNA, ENA and other modifications as described in Wan & Seth 2016 (W Brad Wan, Punit P Seth, Review J Med Chem. 2016 Nov 10;59(21):9645-9667. The Medicinal Chemistry of Therapeutic Oligonucleotides). Thus, the oligonucleotide of the invention preferably comprises one more Locked Nucleic Acid Nucleosides (LNA nucleosides) which are well known 2’- modified nucleosides.
Preferably, the one or more modified nucleosides are (S)-6’-methyl-beta-D-oxy-LNA (ScET) LNA nucleosides. More preferably, the one or more modified nucleosides are beta-D-oxy-LNA nucleosides,
Nucleobase modification include, but are not limited to, 5-methyl-cytosine, pseudo-uridine, 5- Methyluridine, 8-Oxoguanine, 2-thio-thymine, Diaminopurine, abasic nucleosides and others as also described in Brad&Seth 2016 and Robert et al., 2020 (Thomas C Roberts, Robert Langer, Matthew J A Wood. Review Nat Rev Drug Discov. 2020 Oct;19(10):673-694. Advances in oligonucleotide drug delivery) Alternatively or additionally, the chemically modified antisense oligonucleotide contains at least one modified nucleobase. For example, the at least one modified nucleobase is 5 -methylcytosine. Also, the ASO may comprise at least pseudouridine, or at least one 8-oxoguanine as modified nucleobase.
Alternatively or additionally, the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2-O-Methoxyethyl-RNA, 2’-O- Methyl-RNA, 2’-Fluoro-RNA.
In a preferred embodiment, the antisense oligonucleotide of the present invention has a gapmer structure, i.e. is a gapmer. Gapmers are well known in the art. The term refers to (single stranded) DNA antisense oligonucleotide structures with RNA-like segments on both sides (flanking regions). Gapmers bind to the target sequence and down-regulate target gene expression through the induction of RNase H cleavage.
Suitable gapmer designs are well known in the art and are e.g. reviewed in Crooke et al. which herewith is incorporated by reference in its entirety (Stanley T Crooke, Xue-Hai Liang, Brenda F Baker, Rosanne M Crooke. Review J Biol Chem. 2021 Jan-Jun;296. Antisense technology: A review).
Preferably, the gapmer is a LNA gapmer in which the flanking regions comprise LNA nucleosides, such as D-oxy LNA nucleosides. However, the gapmer may also comprise 2’O-Methoxyethyl (MOE) nucleosides in the flanking regions. Such gapmers are frequently referred to as MOE gapmers.
In a preferred embodiment, the antisense oligonucleotide of the present invention has a gapmer structure and at least one modified internucleoside linkage. In a preferred embodiment, the antisense oligonucleotide of the present invention has a gapmer structure and at least 10 modified intemucleoside linkages. In another preferred embodiment, the antisense oligonucleotide of the present invention has a gapmer structure and all linkages are modified intemucleoside linkages. The modified linkages are described herein above. In an embodiment, the modified linkages are phosphorothioate intemucleoside linkages. In another embodiment, the linkages are phosphorodithioates linkages.
In an embodiment, the antisense oligonucleotide of the present invention is a compound selected from the compounds shown in Table Al (see column “Compound”), wherein
• Adx represents 2'deoxyadenosine-3' -phosphorothioate
• Aox represents 2'-O-methyladenosine-3'-phosphorothioate
• Amx represents 2'-O-Methoxyethyladenosine-3' -phosphorothioate
• Alx represents 2'-O-beta-D-oxy LNA adenosine-3' -phosphorothioate
• Cdx represents 2'deoxycytidine-3' -phosphorothioate
• Cox represents 2'-O-methylcytidine-3 '-phosphorothioate
• Edx represents 2'deoxy-5-methylcytidine-3' -phosphorothioate • Emx represents 2'-O-Methoxyethyl-5-methylcytidine-3' -phosphorothioate
• Elx represents 2'-O-beta-D-oxy LNA -5-methylcytidine-3' -phosphorothioate
• Gdx represents 2'deoxyguanosine-3' -phosphorothioate
• Gox represents 2'-O-methylguanosine-3 '-phosphorothioate
• Gmx represents 2'-O-Methoxyethylguanosine-3' -phosphorothioate
• Glx represents 2'-O-beta-D-oxy LNA guanosine-3' -phosphorothioate
• Tdx represents 2'deoxythymidine-3' -phosphorothioate
• Tmx represents 2'-O-Methoxyethylthymidine-3' -phosphorothioate
• Tlx represents 2'-O-beta-D-oxy LNA thymidine-3' -phosphorothioate
• Uo represents 2'-O-methyluridine-3'
• Ad represents 2'deoxyadenosine-3'
• Ao represents 2'-O-methyladenosine-3'
• Am represents 2'-O-Methoxyethyladenosine-3'
• Al represents 2'-O-beta-D-oxy LNA adenosine-3'
• Cd represents 2'deoxycytidine-3'
• Co represents 2'-O-methylcytidine-3'
• Ed represents 2'deoxy-5-methylcytidine-3'
• Em represents 2'-O-Methoxyethyl-5-methylcytidine-3'
• El represents 2'-O-beta-D-oxy LNA -5 -methylcytidine -3'
• Gd represents 2'deoxyguanosine-3'
• Go represents 2'-O-methylguanosine-3'
• Gm represents 2'-O-Methoxyethylguanosine-3'
• G1 represents 2'-O-beta-D-oxy LNA guanosine-3'
• Td represents 2'deoxythymidine-3'
• Tm represents 2'-O-Methoxyethylthymidine-3',
• T1 represents 2'-O-beta-D-oxy LNA thymidine-3', and
• Uo represents 2'-O-methyluridine-3.'
All intemucleoside linkages present in the compounds shown in Table Al are phosphorothioate linkages.
In Table Al, a three letter code or two letter code was used in order to describe the modified nucleotides that are present in the oligonucleotide compounds. Additionally, each of the compounds in Table Al contains a two letter code in the 3’ end, which does not contain the phosphorothioate group. The base and sugar groups of the two letter code is otherwise identical to the three letter. Table A2 provides a translation of the two or three letter codes to their chemical names.
Table A2: Overview on modified nucleotides
Figure imgf000022_0001
Figure imgf000023_0001
Complex Macromolecules, such antisense oligonucleotides according to the present invention with non-natural chemical modifications, can be also depicted in the HELM format (HELM: “Hierarchical Editing Language for Macromolecules”). The HELM format is described in Zhang T, Li H, Xi H, Stanton RV, Rotstein SH. HELM: a hierarchical notation language for complex biomolecule structure representation. J Chem Inf Model. 2012 Oct 22;52(10):2796-806. doi: 10.1021/ci3001925. Epub 2012 Sep 26. PMID: 22947017. The document is herewith incorporated by reference in its entirety. Table A3 shows selected compounds of the present invention in HELM Annotation Format. In a preferred embodiment, the antisense oligonucleotide is a compound as shown in Table A3.
Table A3: Exemplary compounds of the present invention - HELM Annotation Format
Figure imgf000024_0001
Figure imgf000025_0001
Helm Annotation Key for the compounds in Table A3: [LR](G) is a beta-D-oxy-LNA guanine nucleoside,
[LR](T) is a beta-D-oxy-LNA thymine nucleoside,
[LR](A) is a beta-D-oxy-LNA adenine nucleoside,
[LR]([5meC] is a beta-D-oxy-LNA 5-methyl cytosine nucleoside,
[dR](G) is a DNA guanine nucleoside,
[dR](T) is a DNA thymine nucleoside,
[dR](A) is a DNA adenine nucleoside,
[dR](C) is a DNA cytosine nucleoside,
[mR](G) is a 2'-O-methyl RNA guanine nucleoside,
[mR](U) is a 2'-O-methyl RNA uracil nucleoside,
[mR](A) is a 2'-O-methyl RNA adenine nucleoside,
[mR](C) is a 2'-O-methyl RNA cytosine nucleoside,
[sP] is a phosphorothioate internucleoside linkage.
For more details, see also the Helm Annotation Key for Table C3 which also applies to Table A3 (and vice versa).
Preferably, the compound selected from Table Al is a compound which resulted in an efficient downregulation of the target gene in the studies described in Example 1 and/or Example 2.
The results for the experiments in Example 1 are shown in Table 3. In an embodiment, the compound is selected from the compounds resulting in an expression level of NAT8L of 40% or less than 40% , such as of 30% or less than 30% in relation to PBS treated control cells . Information on the expression level can be found in the column “NAT8L PBS norm (A549 High cone)” in Table 3. The results for the experiments in Example 2 are shown in Table 6. In an embodiment, the compound is selected from the compounds resulting in an expression level of NAT8L of 50% or less than 50% , such as of 40% or less than 40% in relation to PBS treated control cells . Information on the expression level can be found in the column “NAT8L PBS norm (HEK293 Low cone)” in Table 6.
In an embodiment, the antisense oligonucleotide is an antisense oligonucleotide with ASO ID 2 17, 2_35, 2_50, 2_51, 2_52, 6_8, 25 113, 29_2, 29_5, 29_6, 29_7, 29_10, 29_23, 29_24, 29_32, 29_34, 29_36, 29_54, 29_70, 29_72, 29_78, 29_79, 29_84, 29_85, 29_86, 29_100, 29_112, 29_121, 29_123, 29_124, 29_125, 29_130, 29_133, 29_138, 29_154, 34_38, 34_39, 34_46, 34_47, 37 15, 38_5, 38 10, 38 11, 51 66 as shown in Table Al in the Examples section.
In preferred embodiment, the antisense oligonucleotide is a compound selected from the compounds shown in Table A3.
In an embodiment, the antisense oligonucleotide is the compound with ASO ID 2 17.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 2 50.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 2 52.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 2 35.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 25 111.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 25 113.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 5.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 10.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 34. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 70. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 78. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 79. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 84. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 85. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 86. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 124. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 121. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 130. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 29 138 In another embodiment, the antisense oligonucleotide is the compound with ASO ID 34 39. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 34 46. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 37 15. In another embodiment, the antisense oligonucleotide is the compound with ASO ID 38 10.
In another embodiment, the antisense oligonucleotide is the compound with ASO ID 51 6.
In another embodiment, the antisense oligonucleotide of the present invention is a compound selected from the compounds shown in Table Cl in Figure 1 (see column “Compound”). In Table Cl, a three letter code or two letter code was used in order to describe the modified nucleotides that are present in the oligonucleotide compounds. Additionally, each of the compounds in Table C 1 contains a two letter code in the 3 ’ end, which does not contain the phosphorothioate group or phosphate. The base and sugar groups of the two letter code is otherwise identical to the three letter. Table C2 provides a translation of the two or three letter codes to their chemical names.
The annotations, typically, also apply to the three or two letter codes of the compounds in Table Al and vice versa. An x between two nucleotides represents a phosphorothioate intemucleoside linkage. An o between two nucleotides represents a phosphodiester linkage.
Table C2: Overview on modified nucleotides
Figure imgf000027_0001
Figure imgf000028_0001
Table C3 shows selected compounds of the present invention in HELM Annotation Format. In a preferred embodiment, the antisense oligonucleotide is a compound as shown in Table C3.
Table C3: Exemplary compounds of the present invention - HELM Annotation Format
Figure imgf000028_0002
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Helm Annotation Key for the compounds in Table C3:
[LR](G) is a beta-D-oxy-LNA guanine nucleoside,
[LR](T) is a beta-D-oxy-LNA thymine nucleoside,
[LR](A) is a beta-D-oxy-LNA adenine nucleoside, [LR]([5meC]) is a beta-D-oxy-LNA 5-methyl cytosine nucleoside,
[MOE](G) is a 2'-O-Methoxy ethyl guanine nucleoside,
[MOE](T) is a 2'-O-Methoxy ethyl thymine nucleoside,
[MOE](A) is a 2'-O-Methoxy ethyl adenine nucleoside, [MOE]([5meC]) is a 2'-O-Methoxyethyl-5-methyl cytosine nucleoside, [mR](G) is a 2'-O-methyl RNA guanine nucleoside,
[mR](U) is a 2'-O-methyl RNA uracil nucleoside,
[mR](A) is a 2'-O-methyl RNA adenine nucleoside,
[mR](C) is a 2'-O-methyl RNA cytosine nucleoside,
[dR](G) is a DNA guanine nucleoside,
[dR](T) is a DNA thymine nucleoside,
[dR](A) is a DNA adenine nucleoside,
[dR](C) is a DNA cytosine nucleoside,
[dR]([5meC]) is a DNA 5-methyl cytosine nucleoside,
[R](G) is a RNA guanine nucleoside,
[R](T) is a RNA thymine nucleoside,
[R](A) is a RNA adenine nucleoside,
[R](C) is a RNA cytosine nucleoside,
[sP] is a phosphorothioate internucleoside linkage
P is a phosphate internucleoside linkage
The number prior to the underline indicates the target ID of the antisense compound. Thus, ASO ID 66 117 binds to a target sequence with Target ID No 66. The compound might also bind to other target IDs of the present invention (as some target IDs overlap, see e.g. Table DI).
Preferably, the compound selected from Table C 1 is a compound which resulted in an efficient downregulation of the target gene in the studies described in Example 5. The results for the experiments are shown in Table 8. Preferably, the compound is selected from the compounds resulting in an expression level of NAT8L of 50% or less than 50% in relation to PBS treated control cells. Preferably, the compound is selected from the compounds resulting in an expression level of NAT8L of 40% or less than 40% in relation to PBS treated control cells.
Preferably, the antisense oligonucleotide (or composition) of the present invention shall be administered to the CNS, in particular to the brain. Accordingly, the ASO is delivered to CNS through intrathecal injection - as it is e.g. the current state of art for similar ASO e.g. Nusinersen/Spinraza. Thus, the ASO of the present invention or the pharmaceutical composition is, preferably, administered intrathecally. In addition to delivery to the CNS, the ASO can be administered by subcutaneous or intravenous administration either with or with or without a conjugate in order to reach the peripheral nervous system, which is also negatively affected by the loss of ASPA expression.
The present invention further relates to a conjugate comprising the antisense oligonucleotide of the present invention and a conjugate moiety. Preferably, the conjugate moiety is covalently bound to the antisense oligonucleotide, e.g. via one or more linker nucleotides, such as one, two, three or four linker nucleotides (or more). The linker may be cleaved after administration to the patient. Preferably, the antisense oligonucleotide of the present invention shall be delivered or administered to the CNS, in particular to the brain. Accordingly, it is envisaged that the conjugate moiety is a moiety that allows the crossing of the conjugate of the blood brain barrier. For example, the moiety can be and antibody or antigen-binding fragment thereof targeting the transferrin receptor.
The antisense oligonucleotides of the present invention can be administered/delivered ‘unassisted’ in saline solution. However, distribution to certain tissues and uptake in cells can be enhanced by conjugates and formulation techniques. Conjugation to ASOs could be, peptides, antibodies and aptamers binding to receptors on target cells or proteins mediating transcytosis e.g. the transferrin receptor. Antisense oligos can also be conjugated to naturally occurring ligands or modifications hereof as exemplified by GalNac conjugation binding with high affinity to asialoglycoprotein receptor 1 (ASGR1, ASPGR) and Alpha-tocopherol conjugation and interaction with transfer protein Alfa-TTP. This could also be small molecules generated through medicinal chemistry with high affinity for known receptors and transporter. Moreover, it could be conjugations to long chained fatty acids that modify the hydrophobicity and protein binding properties of the ASOs, but also could function through their capacity to bind to lipoprotein particles and hence function through the endogenous mechanism for lipid transport and uptake.
In addition to conjugation, tissues delivery and cellular uptake of ASOs of the present invention can be enhanced through formulation with nanocarriers, that facilitates crossing of biological barriers such as cellular membranes. Various types of nanocarriers have been described with with favorable properties for delivery of nucleic acids e.g. lipid nanoparticles (LNPs) as used for BioNTech mRNA vaccines, LNPs functionalizes with peptides, pegylated lipids, cationic lipids, exomes (lipid bilayers) both artificial and natural exosomes such as milk exosomes and spherical nucleic acids and others as described in further details in Roberts et al., 2020 (Thomas C Roberts, Robert Langer, Matthew J A Wood. Review Nat Rev Drug Discov. 2020 Oct;19(10):673-694. Advances in oligonucleotide drug delivery).
The present invention further relates to a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or the conjugate of the present invention.
Typically, a pharmaceutical composition comprises the antisense oligonucleotide or the conjugate of the present invention together with a pharmaceutically acceptable carrier and/or, in particular, a pharmaceutically acceptable excipient. The term "pharmaceutically acceptable", as used herein, refers to the non-toxicity of a material which, in certain exemplary embodiments, does not interact with the action of the oligonucleotide or the conjugate present in the pharmaceutical composition.
The term “carrier”, as used herein, refers to an organic or inorganic component, of a natural or synthetic nature, in which the active component is combined in order to facilitate, enhance or enable application. The term “excipient”, as used herein, is intended to include all substances which may be present in a pharmaceutical composition and which are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surface active agents, preservatives, emulsifiers or buffer substances.
The form of the pharmaceutical composition, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and gender of the patient, etc.
In an embodiment, the pharmaceutical composition can be formulated for intrathecal administration. Thus, the antisense oligonucleotide or conjugate of the present invention is preferably administered by intrathecal administration a route of administration for drugs via an injection into the spinal canal. Thereby, it reaches the cerebrospinal fluid and the brain.
The present invention further relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use in medicine.
Accordingly, the present invention relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use treating Canavan disease.
Further, the present invention relates to the use of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament for treating Canavan disease. Canavan disease, also referred to as Canavan-van Bogaert-Bertrand disease is a degenerative disease that is associated with a progressive damage to nerve cells and loss of white matter in the brain. The disease is inherited in an autosomal recessive manner. It is caused by mutations the ASPA gene which codes for the enzyme aspartoacylase. Decreased aspartoacylase activity prevents the normal breakdown of N-acetyl aspartate.
Accordingly, the present invention relates to the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for use treating Canavan disease.
Further, the present invention relates to the use of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention for the manufacture of a medicament for treating Canavan disease. Further, the present invention relates to a method of treating Canavan disease, comprising administering pharmaceutically effective amount of the antisense oligonucleotide according to the present invention, the conjugate according to the present invention, or the pharmaceutical composition according to the present invention to a subject suffering from Canavan disease.
The term “treating” or “treatment”, as used herein, refers to the administration of a compound or composition or a combination of compounds or compositions to a subject in order to: ameliorate Canavan disease. Thus, the term encompasses both the amelioration of one or more symptoms of the Canavan disease or prevention of the worsening of one or more symptoms, i.e. prophylaxis. The amelioration of symptoms also includes the reduction of one or more symptoms. Thus, the term, preferably, refers to the reduction of one or more symptoms of the disease. In other words, the treatment is typically a disease modifying treatment that reduces one or more symptoms of Canavan disease. In an embodiment, the development of disease pathology is inhibited. It will therefore be recognized that treatment as referred to herein may, in some embodiments, be prophylactic or disease modifying. It is to be understood that the treatment does not allow a complete cure of Canavan disease.
Canavan disease is known to decrease the life expectancy. Thus, the term “treatment” also includes increasing the life expectancy of a subject (as compared to an untreated subject).
In accordance with the present invention, the subject to be treated is a subject suffering from Canavan disease. However, the patient might not yet show symptoms of Canavan disease at the time of the treatment. Typically, the subject shows symptoms of Canavan disease. Symptoms of Canavan disease are well known in the art and include (but are not limited to) one or more of lack of motor development, macrocephaly, lack of head control and abnormal muscle tone. Preferably, the subject has been diagnosed through genetic testing to suffer from Canavan disease. Typically, the disease is diagnosed at the infant age. Also typically, the diagnosis involves genetic testing and/or the detection of increased levels of N-acetylaspartic acid (NAA) in the urine.
The terms “subject” and “patient” are used interchangeably herein. The “subject” or “patient” may be a vertebrate. The term includes both humans and other animals, particularly mammals, and other organisms. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. Preferably, the subject is a human subject suffering from Canavan disease.
The present invention further relates to a method for identifying a candidate compound for the treatment of Canavan disease, comprising a) providing an antisense oligonucleotide according to the present invention, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c). The antisense oligonucleotide is preferably the antisense oligonucleotide of the present invention. Accordingly, it shall comprise a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in the human NAT8Lgene (i.e. mRNA or premRNA). The definitions provided herein above preferably apply mutatis mutandis.
Preferably, said method is an in vitro method. In step a) of the above method of the present invention an antisense oligonucleotide of the present invention is provided. Preferably, the antisense oligonucleotides are complementary to a target region as set forth herein elsewhere. In step b) the antisense oligonucleotide shall be contacted with a host cell. Said host cell shall express the NAT8L gene.
Embodiments of the present invention
In the following, preferred embodiments of the present invention are provided. The definitions and explanations made herein above apply mutatis mutandis.
1. An antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in the human NAT8L (N-acetyltransferase 8 like) gene.
2. The antisense oligonucleotide of embodiment 1, wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 81.
3. The antisense oligonucleotide of embodiment 1 or 2, wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 29.
4. The antisense oligonucleotide of any one of embodiments 1 to 3, wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of: SEQ ID NO: 4 to SEQ ID NO: 10.
5. The antisense oligonucleotide of any one of embodiments 1 to 4, comprising a stretch of at least 12, or at least 14 nucleotides which is at least 90% complementary to said target sequence.
6. The antisense oligonucleotide of any one of embodiments 1 to 5, wherein said stretch is 100% complementary to said target sequence.
7. The antisense oligonucleotide of any one of embodiments 1 to 6, wherein the antisense oligonucleotide has a length of 12 to 30 nucleotides.
8. The antisense oligonucleotide of embodiment 7, wherein the antisense oligonucleotide has a length of 14 to 22 nucleotides, such as a length of 16 to 20 nucleotides.
9. The antisense oligonucleotide of any one of embodiments 1 to 8, wherein the antisense oligonucleotide comprises or consists of a nucleic acid sequence as shown in SEQ ID NO: 403 to SEQ ID NO: 723. The antisense oligonucleotide of any one of embodiments 1 to 9, wherein the antisense oligonucleotide is capable of reducing the amount of NAT8L (N-acetyltransferase 8 like) mRNA in a host cell expressing said NAT8L mRNA. The antisense oligonucleotide embodiment 10, wherein the target cell is a human cell, such as a cell of the CNS. The antisense oligonucleotide of any one of embodiments 1 to 11, wherein the antisense oligonucleotide is a chemically modified antisense oligonucleotide. The antisense oligonucleotide of embodiment 12, wherein the chemically modified antisense oligonucleotide contains one or more modified nucleosides. The antisense oligonucleotide of embodiment 13, wherein the one or more modified nucleosides, is a sugar modified nucleoside, such as a 2’ sugar modified nucleoside. The antisense oligonucleotide of any one of embodiments 11 to 14, wherein the chemically modified antisense oligonucleotide contains at least one modified nucleobase. The antisense oligonucleotide of embodiment 13, wherein the at least one modified nucleobase is 5 -methylcytosine. The antisense oligonucleotide of any one of embodiments 11 to 14, wherein the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2’-O-Methoxyethyl-RNA, 2’-O-Methyl-RNA, 2’-Fluoro-RNA. The antisense oligonucleotide of any one of embodiments 1 to 17, wherein the antisense oligonucleotide comprises at least one modified intemucleoside linkage, such as at least one Phosphorothioate intemucleoside linkage, at least one Phosphorodithioate intemucleoside linkage, at least one Phophoroamidate intemucleoside linkage, at least one methyl phosphonate intemucleoside linkage, at least one phosphotriester intemucleoside linkage, at least one boranophosphate intemucleoside linkage or at least one phosphoryl guanidine intemucleoside linkage. The antisense oligonucleotide of embodiment 18, wherein all intemucleoside linkages are modified intemucleoside linkages, such as Phosphorothioate intemucleoside linkages. The antisense oligonucleotide of any one of embodiments 1 to 19, wherein the antisense oligonucleotide comprises one or more modified nucleosides being LNA (locked nucleic acid) nucleosides, The antisense oligonucleotide of embodiment 20, wherein the LNA nucleoside(s) is (are) a beta- D-oxy LNA nucleosides. The antisense oligonucleotide of any one of embodiments 1 to 21, wherein the antisense oligonucleotide has a gapmer structure. The antisense oligonucleotide of any one of embodiments 1 to 22, wherein the antisense oligonucleotide is a compound shown in Table Al, such as a compound as shown in Table A3. A conjugate comprising the antisense oligonucleotide of any one of embodiments 1 to 23, wherein a conjugate moiety is covalently bound to the antisense oligonucleotide. 25. A pharmaceutical composition comprising the antisense oligonucleotide of any one of embodiments 1 to 23 or the conjugate of embodiment 24.
26. The antisense oligonucleotide of any one of embodiments 1 to 23, the conjugate of embodiment 24, or the pharmaceutical composition of embodiment 25 for use in treating Canavan disease.
27. A method for identifying a candidate compound for the treatment of Canavan disease, comprising a) providing an antisense oligonucleotide as defined in any one of embodiments 1 to 23, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
All patents, patent applications, and publications or public disclosures referred to or cited herein are incorporated by reference in their entirety.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
EXAMPLES
Oligonucleotide synthesis
Oligonucleotide synthesis is generally known in the art and can be acquired from multiple different providers. In the listed in vitro experiments (example 1, 2, 3, 5, 6 and 7) all oligonucleotides were acquired from Biosearch Technologies (Lystrup, Denmark). In brief, after cleavage from the solid support the oligonucleotides are cartridge purified using Ammonium acetate; Dissolved to 750pM in PBS and purity determined by LC/MS (> 80%).
In the listed in vivo experiments (example 9 and 10) the oligonucleotides were acquired from WuXi AppTec (China). After cleavage from the solid support the oligonucleotides were HPLC-purified to >90% purity (LC-TOF/MS) and lyophylized as sodium salt followed by formulation in sterile 0,9 % sodiumchloride. Endotoxin test were performed (Kinetic turbidimetric or chromogenic LAL assay) to ensure endotoxin levels <1.0 EU/mg.
The produced compounds are shown in Table Al and Table Cl (see Figures 1 and 2). All compounds shown in Table Al were tested in A549 cells (see Example 1) and/or in HEK293 cells (see Example 2 and example 4). All compounds shown in Table Cl were tested in HEK293 cells (see Example 5). Some of the ASO compounds which led to an efficient downregulation of NAT8L were further tested for potency in HEK cells and iPSC derived neurons as well as for in vivo acute tolerability in mice and for pharmacodynamics and tolerability in non-human primates, Example 1: Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in A549 cells at single test concentration.
In order to interrogate the efficacy of the designed and synthesized ASOs a cell-based screening assay was developed using cells with an endogenous expression of NAT8L premRNA. In that optimization process various cell densities and compound incubation periods and concentrations were tested before reaching the assay conditions as described below.
The A549 cells were maintained and expanded as recommended by the supplier (ECACC, acquired from Merck, 86012804-1VL). The cells were grown to 70-80% confluency, the cells are then trypsinized and resuspended in growth media. Viable cells are counted using trypan blue and a Countess 3 automatic cell counter. The appropriate number of cells are diluted in complete growth media, mixed by gentle pipetting, added to reagent reservoirs, and distributed into 96-well plates using a multichannel pipette in a total volume of 195 pl/well. Sterile PBS is added to the moats of the 96-well culture plates (Nunc™ Edge™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplates) to reduce evaporation and potential plate effect. After 24 h of incubation the NAT8L ASOs (Table 3, see columns “ASO ID”, the compounds are described in Table Al), are added directly to the growth media from a 20-Fold stock dilution in PBS reaching a final ASO concentration of 10 pM. Table 1 summarizes the most important parameters relating to the cellular work.
Table 1: Information on cell line A549
Figure imgf000041_0001
In addition to the ASOs designed to target NAT8L each plate also included 7 PBS controls (5 pL) positive a positive control ASOs (N=2, pr. plate) targeting the ATXN3 gene, moreover 30 non-targeting gapmer ASOs were used as negative controls distributed across randomly across all screening plates to monitor possible false positives.
After 48 hours, cells were harvested by gently aspirating and RNA was extracted using the Macher ey-Nagel NucleoSpin 96 RNA Kit, according to the manufactures instructions and eluted in 75 pl of water. Before qPCR based expression analysis 10 pl of the RNA containing eluate is transferred to a new plate and diluted 10-fold in RNAse free water, then heat chocked at 90°C for 40 s and placed on ice. The diluted and heat shocked RNA is used as input template for the qPCR, using qScript™ XLT One-Step RT-qPCR ToughMix® (cat# 95134-500) from QuantaBio and qPCR assays from Integrated DNA technologies (IDT) listed in Table 2.
Table 2: qPCR assay used for screening NAT8L targeting ASOs.
Figure imgf000042_0001
The QPCR reaction was run in 384 wells using a QuantStudio 7 Flex (applied biosystems by Thermo Fisher Scientific. Quantities of NAT8L mRNA was calculated applying the ddCT method and using the median of all the PBS treated wells within the same plate as the ‘untreated control’.
The expression level of NAT8L following ASO treatment (Table 3) is thus shown as percent of the PBS-treated wells.
Altogether the screen identified a number of different ASOs with high level of KD (such as more than 70%) targeting various regions of the NAT8L premRNA. These sequences and their target sequences are of particular interest for the further optimization of our compounds.
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Example 2 Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in HEK cells at single test concentration.
Based on screening results from example 1, more ASOs were designed and synthesized. The majority of these were designed to target in or vicinity of NAT8L sequences that had been identified in example 1 as being target sequences where high efficacy ASOs could be generated. To better identify the most potent and efficacious compounds lower concentration of compounds were used (1 pM) than in the first screen (10 pM) in example 1. Moreover, a new cell-based screening assay was developed in HEK293 cells as described below.
The HEK293 cells were maintained and expanded as recommended by the supplier. The cells were grown to 70-80% confluency, the cells are then trypsinized and resuspended in growth media. Viable cells are counted using trypan blue and Vi-CELL automatic cell counter (Beckman Coulter). The appropriate number of cells are diluted in complete growth media, mixed by gentle pipetting, added to reagent reservoirs, and distributed into 96-well plates using a multichannel pipette in a total volume of 190 pl/well. Sterile PBS is added to the moats of the 96-well culture plates (Nunc™ Edge™ 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplates) to reduce evaporation and potential plate effect. After 24 h of incubation the NAT8L ASOs (Table 6, see columns “ASO ID”), are added directly to the growth media from a 20-Fold stock dilution in PBS reaching a final ASO concentration of 1 pM. Table 4 summarizes the most important parameters relating to the cellular work.
Table 4: Information on HEK293 cells
Figure imgf000048_0001
In addition to the ASOs designed to target NAT8L each plate also included 10 PBS controls (10 pL), 4 NAT8L positive control ASOs and two ASO controls targeting the ATXN3 gene. After 120 hours, cells were harvested by gently aspirating and RNA was extracted using the Macher ey-Nagel NucleoSpin 96 RNA Kit, according to the manufactures instructions and eluted in 75 pl of water. qPCR experiments were carried out as described in Example 1. Quantities of NAT8L mRNA was calculated applying the ddCT method and using the median of all the PBS treated wells within the same plate as the ‘untreated control’. The expression level of NAT8L following ASO treatment (Table 6) is thus shown as percent of the PBS-treated wells.
Altogether, the data shows that additional high efficacy compounds can be generated in previous identified target sites (Example 1) and in vicinity of these target sites. Table 6: Expression level of NAT8L in relation to PBS treated control cells (in %)
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Example 3: Determination of IC50 values of NAT8L targeting ASOs in HEK cells
To validate the hits identified in single concentration screens (Example 1 and 2) and to rank compounds on their potency a concentration response experiments were subsequently carried out for compounds showing high level of knock down.
Using the same method as describe in example 2 but incubating the cells with different concentrations of ASO (0,01; 0,0316; 1; 0,316; 1,0; 3,16; 10; 31,6 pM) allowed the generation of concentration response curves and IC50 values.
Concentration response curves were generated using the GraphPad prism software version9 using the “log(inhibitor) vs. response — Variable slope (four parameters)” fit with bottom constrained to >0 and top=100. IC50 values are shown in Table 7.
Table 7: IC50 level of selected compounds
Figure imgf000054_0001
Figure imgf000055_0001
In conclusion, the concentration response experiments validated the hits identified from single concentration screen in example 1 and 2 and showed that highly potent compounds have been identified.
Example 4: Overview on identified target regions within the NAT8L pre-mRNA sequence (SEQ ID NO: 1) which allow for efficiently downregulating NAT8L Target sequences that were found in the studies underlying the present invention are shown in Table Bl.
Figure imgf000055_0002
Figure imgf000056_0001
Figure imgf000057_0001
Targeting sequences as shown in Table B2 allowed for a very efficient down-regulation of the target gene. Table B2: Target sequences
Figure imgf000057_0002
Figure imgf000058_0001
The best results were obtained when ASOs were used that are complementary to target sequences shown in Table B3.
Figure imgf000058_0002
Example 5. Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in HEK cells
Further ASOs were designed and synthesized. The newly synthesized are shown in Table Cl. The in vitro efficacy of antisense oligonucleotides was tested as described in Example 2. The results are shown in Table 8 which shows the expression level of NAT8L following ASO treatment as percent of the PBS-treated wells. Altogether, the data shows that additional high efficacy compounds can be generated in previous identified target sites and in vicinity of these target sites. Further, high efficacy compounds were generated for new target sequences (e.g. target sequences with Targed ID 53, 56, 58, 59, 63, and 67, see Table DI in Example 10.
able 8: The table shows the expression level of NAT8L relative to untreated control (UTC%). The header of each column refers to the ell line (e.g. HEK293, A549), then the experiment number of each independent experiment (e.g. 1, 2 and 3 and 4), and the concentration of
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Example 6: Determination of IC50 values of NAT8L targeting ASOs in HEK cells To validate the hits identified in single concentration screens (Example 5) and to rank compounds on their potency a concentration response experiments were subsequently carried out for compounds showing high level of knock down.
Using the same method as describe in example 2 but incubating the cells with different concentrations of ASO (0,01; 0,0316; 1; 0,316; 1,0; 3,16; 10; 31,6 pM) allowed the generation of concentration response curves and determination of IC50 values.
Concentration response curves were generated using the GraphPad prism software version9 using the “log(inhibitor) vs. response — Variable slope (four parameters)” fit with bottom constrained to >0 and top=100. IC50 values are shown in Table 9.
Table 9: IC50 value of selected compounds
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Example 7. Testing in vitro efficacy of antisense oligonucleotides targeting NAT8L in iPSC derived human neurons at multiple test concentration.
A set of ASOs selected to cover most of the preferred regions were tested in iPSC derived human neurons to confirm activity and potency of the most active ASOs in a relevant cellular model.
The iPSC derived neurons were maintained as recommended by the supplier (Fuji film, 01279). 96-well cell culture plates (Nunc™ Edge™ 96-Well, Nunclon Delta-Treated, Flat- Bottom Microplates) were coated with sterile 0,07% polyethylenimide for 1 hour, washed with PBS and water and coated with 0,01 mg/ml of laminin for 1 hour. The laminin solution was removed with no further washing of the culture plates and iPSC derived neurons were thawed and seated at a density of 80.000 neurons pr well as described in Fuji films: iCell GlutaNeurons User's Guide in a total volume of 190pl/well plates using a multichannel pipette. After 24 and 96 hours of incubation, half of the media was changed. 96 hours after seeding NAT8L ASOs (Table 11, see columns “ASO ID”), are added directly to the growth media from a 20-Fold stock dilution in PBS reaching a final ASO concentration of either 0,2, 1, 5 or 25 pM. Table 10 summarizes the most important parameters relating to the cellular work.
Table 10: Information on iPSC derived neurons
Figure imgf000070_0002
Neurons were harvested after 96 hours treatment with ASO by gently aspirating the growth media, addition of 125 pl RLT buffer and RNA was extracted using the RNeasy 96, QIAcube HT kit (Qiagen #74171) according to the manufactures instructions and eluted in 75 pl of water. qPCR experiments were carried out as described in Example 1. Quantities of NAT8L mRNA was calculated applying the ddCT method and using the median of all the PBS treated wells within the same plate as the ‘untreated control’. The expression level of NAT8L following ASO treatment (Table 11) is thus shown as percent of the PBS-treated wells. As for example 3, concentration response curves were generated using the GraphPad prism software version9 using the “log(inhibitor) vs. response — Variable slope (four parameters)” fit with bottom constrained to >0 and top=100. IC50 values are shown in Table 11.
Table 11: Expression levels of human NAT8L in iPSC derived neurons shown as %UnTreatedControl (%UTC) and as IC50 Value
Figure imgf000071_0001
Example 8 - In vivo acute toxicity in mice of ASOs targeting NAT8L.
To test the acute toxicity potential of the NAT8L targeting ASOs, acute in vivo CNS toxicity study was tested by neurob ehavi oral scoring after intracerebroventricular dosing, as described below.
Experimental mice. In vivo acute tolerability of the antisense oligonucleotides were tested in mice, with 6 mice per ASO group. Mice at 8-10 weeks of age aged were housed in European IVC cages type IIL with TAPVEI aspen bedding (Tapvei Eatonis Oil, Estonia). The cages were enriched with nesting material, wooden sticks and hiding material. The light cycle was 12-hour dark and 12-hour light. Diet was pelleted complete diet (Altromin 1324, Brogaarden), and municipal drinking water. Diet and water were administered ad libitum.
All animals were inspected on daily basis for their general health condition. Any clinical signs or behavioral abnormalities was recorded. Humane endpoints and premature termination: Any animal showing clinical signs of moderate pain or moderate distress, or any degree of suffering was handled as appropriate, as discontinuation of the administration of test articles or euthanizing of the animal. Animals exhibiting clinical signs were humanely euthanized if they exceeded the limits of the study specific humane endpoints according to the European and Danish legislation on animals in experimentation.
Administration by ICV injection:Pre-dosing analgesia was given at least 30 min before dosing is initiated, all animals received preventive pain treatment with Meloxicam (2 mg/kg SC). The mice were be anesthetized with isoflurane before dosing. For injection, the G23 needle was mounted on a stand so it precisely penetrated 3.9 mm through the mouse's skull. The dose volume of 5 pL was injected over 30 seconds and the animal was then placed back in its cage and clinically observed according to the instructions below.
Following dosing, the animals were observed closely. The assessment was performed according to a scoring scheme where a score of 0 to 4 was given for the following five parameters: activity level (increased or decreased activity, respectively), motor function, posture/ presentation, and muscle tremors/cramps as outlined in table 12. Scoring was performed before dose, 30 min and 60 min after dosing.
Table 12:
Figure imgf000072_0001
Average neurob ehavi oral scoring at 1 hour post dose for a 50 pg dose or 100 pg dose is summarized in table 13. If the mice had to be euthanized before 1 hour post dosing, they got a score of 20.
Table 13: Acute neurotoxicity data
Figure imgf000073_0001
The generated acute toxicity data in mice shows that the observed acute toxicity is highly dependent on the ASO sequence. Some ASOs e.g. 29 138 and 34 46 are highly neurotoxic and giving rise to toxic effects of a severity requiring the mice to be euthanized shortly after dosing. In contrast ASOs such as 29_79, 29_84, 29_85, 29_124 and 66_36 showed only minor short lasting neurological signs. Example 9. Single dose in vivo efficacy test in African green monkeys (Chlorocebus sabaeus) To evaluate the efficacy of one of the most preferred ASOs; ASO ID 29 85 in vivo using a relevant dosing paradigm and in a species of high translational value, a single dose of ASO was delivered to African green monkeys via an IT-catheter followed by a two week in life phase before harvest of brain tissues.
After a 4-week recovery from IT CSF access port implantation, monkeys were fasted overnight, sedated with ketamine/xylazine and placed in a prone position. 0,5 ml of ASO or saline were injected followed by a saline flush of 0.5 mL/kg monkey.
2 monkeys were dosed with ASO ID 29_85 and 2 monkeys were dosed with 0,9% saline. Following dosing the monkey were monitored closely for their general wellbeing throughout the 2 weeks of the experiment, without observing any adverse effects.
At termination, monkeys were sedated intramuscularly with ketamine and xylazine to effect and euthanized with sodium pentobarbital. The following brain tissues was collected with a 2 mm punch into DNase/RNase free cryotubes and snap-frozen: Frontal Cortex, Parietal Cortex, Occipital Cortex, Ventral Striatum (nucleus accumbens area), Hippocampus, Ventral Pons, Medulla, Cerebellum, Ventral Midbrain, Cervical Spinal Cord, Thoracic Spinal Cord, Lumbar Spinal Cord.
Brain tissue pieces were homogenized in RLT buffer using the Precellus system, then Trizol/choroform extracted and RNA from the aqueous phases were purified on a QIAcube HT using the RNeasy 96, QIAcube HT kit (Qiagen #74171). qPCR was performed as described in example 1 using qPCR assays specific for monkey NAT8L and monkey UBE2D2 for normalization. The reduction is presented as relative reduction compared to the median for saline treated NHPs with 2 punctures from each brain region.
A single 12 mg dose of ASO ID 29 85 administered intrathecally shows significant reduction of NAT8L relative to UBE2D2 in various brain regions as seen in table 14:
Table 14: %NAT8L mRNA levels (Normalized to UBE2D2) of saline treated NHP "average±stdev).
Figure imgf000074_0001
Altogether the data shows that ASOs identified in our screening cascade consisting of in vitro efficacy in cell lines (examplel-6), human IPSC derived neurons (example 7) mouse acute toxicity (example 8) can generate ASOs that are well tolerate and highly efficacious in non-human-primates. Example 10: Overview on target regions within the NAT8L pre-mRNA sequence (SEQ ID NO: 1) identified in Example 5.
Target sequences of the invention that were found in Example 5 are shown in Table DI. Targeting these regions allows for an efficient downregulation of the NAT8L gene. Some of the identified target sequences, in principle, correspond to some of the target sequences described in Table Bl and are subregions or expansions of these target sequences. For more details, see the Table in the specification. Table DI: Target sequences
Figure imgf000075_0001
Targeting sequences as shown in Table D2 allowed for a very efficient down-regulation of the target gene. Table D2: Target sequences
Figure imgf000076_0001
The best results were obtained when ASOs were used that are complementary to target sequences shown in Table D3.
Table D3: Target sequences
Figure imgf000076_0002
Table D4 shows target sequences of preferred compounds.
Table D4: Target sequences of preferred compounds
Figure imgf000076_0003
Figure imgf000077_0002
*can be the target sequences of other preferred compounds as well.
Figure imgf000077_0001

Claims

Claims An antisense oligonucleotide comprising a stretch of at least 10 nucleotides which is at least 90% complementary to a target sequence in the human NAT8L (N- acetyltransferase 8 like) gene. The antisense oligonucleotide of claim 1, wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 58, SEQ ID NO: 4 to SEQ ID NO: 57, SEQ ID NO: 59 to SEQ ID NO: 81, and SEQ ID NO: 882 to 906. The antisense oligonucleotide of claim 1 or 2, wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of SEQ ID NO: 4 to SEQ ID NO: 29, in particular wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence selected from the group of target sequences consisting of: SEQ ID NO: 4 to SEQ ID NO: 10. The antisense oligonucleotide of claim 1 or 2, wherein the stretch of at least 10 nucleotides is at least 90% complementary to a target sequence comprising a sequence as shown in SEQ ID NO: 883, 892, 894, 895, 896, 897, 898, 899, in particular as shown in SEQ ID NO NO: 883, 894, 895, 897, 898 and 899. The antisense oligonucleotide of any one of claims 1 to 4, comprising a stretch of at least 12, or in particular at least 14 nucleotides which is at least 90% complementary to said target sequence. The antisense oligonucleotide of any one of claims 1 to 5, wherein said stretch is 100% complementary to said target sequence. The antisense oligonucleotide of any one of claims 1 to 6, wherein the antisense oligonucleotide has a length of 12 to 30 nucleotides. The antisense oligonucleotide of claim 7, wherein the antisense oligonucleotide has a length of 14 to 22 nucleotides, such as a length of 16 to 20 nucleotides. The antisense oligonucleotide of any one of claims 1 to 8, wherein the antisense oligonucleotide comprises or consists of a nucleic acid sequence as shown in SEQ ID NO: 403 to SEQ ID NO: 881. The antisense oligonucleotide of any one of claims 1 to 9, wherein the antisense oligonucleotide is capable of reducing the amount of NAT8L (N-acetyltransferase 8 like) mRNA in a host cell expressing said NAT8L mRNA. The antisense oligonucleotide claim 10, wherein the target cell is a human cell, such as a cell of the CNS. The antisense oligonucleotide of any one of claims 1 to 11, wherein the antisense oligonucleotide is a chemically modified antisense oligonucleotide. The antisense oligonucleotide of claim 12, wherein the chemically modified antisense oligonucleotide contains one or more modified nucleosides. The antisense oligonucleotide of claim 13, wherein the one or more modified nucleosides, is a sugar modified nucleoside, such as a 2’ sugar modified nucleoside. The antisense oligonucleotide of any one of claims 11 to 14, wherein the chemically modified antisense oligonucleotide contains at least one modified nucleobase. The antisense oligonucleotide of claim 13, wherein the at least one modified nucleobase is 5-methylcytosine. The antisense oligonucleotide of any one of claims 11 to 14, wherein the chemically modified antisense oligonucleotide comprises at least one modified nucleoside selected from the group consisting of: 2’-O-Methoxyethyl-RNA, 2’-O-Methyl-RNA, 2’-Fluoro- RNA. The antisense oligonucleotide of any one of claims 1 to 17, wherein the antisense oligonucleotide comprises at least one modified intemucleoside linkage, such as at least one Phosphorothioate internucleoside linkage, at least one Phosphorodithioate internucleoside linkage, at least one Phophoroamidate internucleoside linkage, at least one methyl phosphonate internucleoside linkage, at least one phosphotriester internucleoside linkage, at least one boranophosphate intemucleoside linkage or at least one phosphoryl guanidine intemucleoside linkage. The antisense oligonucleotide of claim 18, wherein all internucleoside linkages are modified internucleoside linkages, such as Phosphorothioate internucleoside linkages. The antisense oligonucleotide of any one of claims 1 to 19, wherein the antisense oligonucleotide comprises one or more modified nucleosides being LNA (locked nucleic acid) nucleosides, The antisense oligonucleotide of claim 20, wherein the LNA nucleoside(s) is (are) a beta-D-oxy LNA nucleosides. The antisense oligonucleotide of any one of claims 1 to 21, wherein the antisense oligonucleotide has a gapmer structure. The antisense oligonucleotide of any one of claims 1 to 22, wherein the antisense oligonucleotide is a compound shown in Table AL The antisense oligonucleotide of claim 23, wherein the antisense oligonucleotide is a compound as shown in Table A3. The antisense oligonucleotide of any one of claims 1 to 22, wherein the antisense oligonucleotide is a compound shown in Table CL The antisense oligonucleotide of claim 23, wherein the antisense oligonucleotide is a compound as shown in Table C3. The antisense oligonucleotide of any one of claims 1 to 26, wherein the antisense oligonucleotide is a compound with ASO ID 2_17, 29_124, 29_79, 29_84, 29_85, 29_86, 54_2, 54_3, 66_124, 66_126, 66_130, 66_134, 66 135, 66_140, 66_149, 66 160, 66_173, 66_174, 66_176, 66_177, 66 181, 66_182, 66_183, 66_185, 66_188, 66_220, 66_27, 66_36, 66_39, 66_408, 66_47, 66_485, 66_496, 66_544, 66_545, 66_547, 66_567, 66_573, 66_576, 66_584, 66_587, 66_588, 66_592, 66_593, 66_600, 66 63 or 66 64. A conjugate comprising the antisense oligonucleotide of any one of claims 1 to 27, wherein a conjugate moiety is covalently bound to the antisense oligonucleotide. A pharmaceutical composition comprising the antisense oligonucleotide of any one of claims 1 to 27 or the conjugate of claim 28. The antisense oligonucleotide of any one of any one of the preceding claims, the conjugate of claim 28, or the pharmaceutical composition of claim 29 for use in treating
Canavan disease. A method for identifying a candidate compound for the treatment of Canavan disease, comprising a) providing an antisense oligonucleotide as defined in any one of claims 1 to 23, b) contacting a host cell expressing NAT8L mRNA with said antisense oligonucleotide, c) determining the amount of NAT8L mRNA in the said host cell, and d) identifying a candidate compound based on the results of step c).
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